Antibodies to Programmed Cell Death Protein 1

ABSTRACT

Antibodies and antigen binding fragments thereof are provided that immunospecifically bind to PD-1, preferably human or mouse PD-1, and induce or promote an immune response that activates immune cell proliferation or activity. Contrary to the existing paradigm that PD-1 exclusively promotes a suppressive immune response, the disclosed antibodies and antigen binding fragments thereof, immunospecifically bind to PD-1 and cause an activating signal to be delivered to the immune cell that activates the immune cell rather than suppressing the immune cell. In one embodiment, the disclosed antibodies and antigen binding fragments thereof specifically bind to PD-1 expressed on immune cells. The binding of the disclosed antibodies and antigen binding fragments thereof to PD-1 on immune cells causes an activating signal to be transmitted into the immune cell, for example a signal that enhances or promotes cytokine production and/or activation of immune cell proliferation. Immune cells that express PD-1, include but are not limited to B and T cells as well as myeloid-derived cells. In one embodiment, the immune cell is a T cell, preferably a CD8+ T cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/645,289, filed on Mar. 6, 2020, which is a 371 Application ofInternational Patent Application No. PCT/US2018/049,854, filed on Sep.7, 2018, and claims benefit of and priority to U.S. ProvisionalApplication Nos. 62/555,156 filed on Sep. 7, 2017, 62/624,843 filed onFeb. 1, 2018, and 62/657,323 filed on Apr. 13, 2018, all of which areincorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted on Sep. 6, 2018, as a text file named“064466.071USCON sequence listing_ST25.txt” created on Aug. 21, 2018,and having a size of 55.2 kilobytes is hereby incorporated by referencepursuant to 37 C.F.R. § 1.52(e)(5).

TECHNICAL FIELD OF THE INVENTION

The invention is generally related to immunomodulation and to antibodiesthat specifically bind to PD-1 and methods of their use.

BACKGROUND OF THE INVENTION

The programmed cell death receptor protein (PD-1)/programmed cell deathreceptor protein ligand 1 (PD-L1) pathway has shown promising clinicalsuccess as a cancer immunotherapy target. Current antibodies that targeteither PD-1 or PD-L1 can block this interaction and boost the immuneresponse against cancer cells. Successful clinical trials with PD-1monoclonal antibodies and other immune-checkpoint inhibitors have openednew avenues in cancer immunology. However, the failure of a large subsetof cancer patients to respond to new immunotherapies has led tointensified research on combination therapies and predictive biomarkers(Iwai, Y., et al., Journal of Biomedical Science, 24:26 (2017)).

Thus, it is an object of the invention to provide compositions andmethods for modulating PD-1 signal transduction.

It is another object of the invention to provide antibodies and antigenbinding fragments thereof that specifically bind to PD-1 and modulatePD-1 signal transduction.

It is another object of the invention to provide compositions andmethods for treating cancer.

It is another object of the invention to provide compositions andmethods for treating infections.

SUMMARY OF THE INVENTION

Antibodies and antigen binding fragments thereof are provided thatimmunospecifically bind to PD-1, preferably human or mouse PD-1, andinduce or promote an immune response that activates immune cellproliferation or activity. In one embodiment, the disclosed antibodiesand antigen binding fragments thereof specifically bind to PD-1expressed on immune cells. The binding of the disclosed antibodies andantigen binding fragments thereof to PD-1 on immune cells causes anactivating signal to be transmitted into the immune cell, for example asignal that enhances or promotes cytokine production and/or activationof immune cell proliferation. Immune cells that express PD-1, includebut are not limited to B and T cells as well as myeloid-derived cells(Riley, J., Immunol Rev. 229(1):114-125 (2009)). In one embodiment, theimmune cell is a T cell, preferably a CD8+ T cell.

Another embodiment provides a method of stimulating, promoting, orenhancing an adaptive immune response in a subject in need thereof byadministering to the subject an effective amount of the disclosedanti-PD-1 antibodies or an antigen binding fragment thereof to induce,enhance, or promote an adaptive immune response in the subject.

One embodiment provides an antibody or antigen binding fragment thereofhaving heavy chain complementarity-determining regions (CDRs) havingamino acid sequences according to SEQ ID NOs:6, 7, and 8, and lightchain CDRs having amino acid sequences according to SEQ ID NOs:12, 13,and 14, wherein the antibody or antigen binding fragment thereofimmunospecifically binds PD-1.

One embodiment provides an antibody or antigen binding fragment thereofhaving a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%,99%, or 100% sequence identity to SEQ ID NO:4 or 5.

One embodiment provides an antibody or antigen binding fragment thereofhaving a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%,99%, or 100% sequence identity to SEQ ID NO:10 or 11.

One embodiment provides an antibody or antigen binding fragment thereofany having a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%,95%, 99%, or 100% sequence identity to SEQ ID NO:4 or 5 and a lightchain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100%sequence identity to SEQ ID NO:10 or 11.

One embodiment provides a transgenic animal engineered to express anyone of the disclosed antibodies or antigen binding fragments thereof. Inone embodiment, the animal is a mouse.

One embodiment provides a nucleic acid encoding a heavy chain with atleast 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identityto SEQ ID NO:4 or 5.

One embodiment provides a nucleic acid encoding a light chain with atleast 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identityto SEQ ID NO:10 or 11.

One embodiment provides an antibody or antigen binding fragment thereofhaving heavy chain CDRs having amino acid sequences according to SEQ IDNOs:18, 19, and 20, and light chain CDRs having amino acid sequencesaccording to SEQ ID NOs:24, 13, and 25.

One embodiment provides an antibody or antigen binding fragment thereofhaving a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%,99%, or 100% sequence identity to SEQ ID NO:16 or 17.

One embodiment provides an antibody or antigen binding fragment thereofhaving a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%,99%, or 100% sequence identity to SEQ ID NO:22 or 23.

One embodiment provides an antibody or antigen binding fragment thereofhaving a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%,99%, or 100% sequence identity to SEQ ID NO:16 or 17 and a light chainwith at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequenceidentity to SEQ ID NO:22 or 23.

One embodiment provides a nucleic acid encoding a heavy chain with atleast 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identityto SEQ ID NO:16 or 17.

One embodiment provides a nucleic acid encoding a light chain with atleast 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identityto SEQ ID NO:22 or 23.

One embodiment provides an antibody or antigen binding fragment thereofhaving heavy chain CDRs having amino acid sequences according to SEQ IDNOs:29, 30, and 31, and light chain CDRs having amino acid sequencesaccording to SEQ ID NOs:35, 36, and 37.

One embodiment provides an antibody or antigen binding fragment thereofhaving a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%,99%, or 100% sequence identity to SEQ ID NO:27 or 28.

One embodiment provides an antibody or antigen binding fragment thereofhaving a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%,99%, or 100% sequence identity to SEQ ID NO:33 or 34.

One embodiment provides an antibody or antigen binding fragment thereofhaving a heavy chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%,99%, or 100% sequence identity to SEQ ID NO:27 or 28 and a light chainwith at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequenceidentity to SEQ ID NO:33 or 34.

One embodiment provides a nucleic acid encoding a heavy chain with atleast 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identityto SEQ ID NO:27 or 28.

One embodiment provides a nucleic acid encoding a light chain with atleast 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identityto SEQ ID NO:33 or 34.

One embodiment provides an antibody, or antigen binding fragment thereofcontaining three light chain CDRs with amino acid sequences that areselected from the group consisting of SEQ ID NOs: 12, 13, 14, 24, 25,35, 36, or 37.

Another embodiment provides an antibody, or antigen binding fragmentthereof containing three heavy chain CDRs with amino acid sequences thatare selected from the group consisting of SEQ ID NOs: 6, 7, 8, 18, 19,20, 29, 30, or 31.

Another embodiment provides an antibody, or antigen binding fragmentthereof containing three light chain CDRs with amino acid sequences thatare selected from the group consisting of SEQ ID NOs: 12, 13, 14, 24,25, 35, 36, or 37, and three heavy chain CDRs with amino acid sequencesthat are selected from the group consisting of SEQ ID NOs: 6, 7, 8, 18,19, 20, 29, 30, or 31.

One embodiment provides an antibody or epitope binding fragment thereofor a fusion protein that immunospecifically binds to SEQ ID NO:38. Inone embodiment the antibody binds to SEQ ID NO:38 on PD-1. In oneembodiment, the antibody binds to PD-1 expressed on the surface of animmune cell and induces or promotes a signal through PD-1 that activatesor stimulates the immune cell. In one embodiment the immune cell that isactivated or stimulated is a T cell, for example a CD8⁺ T cell.

In some embodiments, the antibody or antigen binding fragment thereof ishuman, mouse, chimeric, humanized, monoclonal, bispecific, trispecificor multispecific.

One embodiment provides a pharmaceutical composition including one ormore of the disclosed antibodies or antigen binding fragments thereof.In some embodiments the pharmaceutical compositions include a secondtherapeutic agent and/or a pharmaceutically acceptable excipient. Anexemplary second therapeutic agent includes cyclophosphamide.

One embodiment provides a method of inducing, promoting, or enhancing animmune response in a subject in need thereof by administering to thesubject an effective amount of one or more of the disclosed antibodiesor antigen binding fragments thereof to induce, promote, or enhance animmune response in the subject.

One embodiment provides a method for treating cancer in a subject inneed thereof by administering to the subject an effective amount of oneor more of the disclosed antibodies or antigen binding fragments thereofto treat cancer in the subject.

One embodiment provides a method for reducing tumor burden in a subjectin need thereof by administering to the subject an effective amount ofone or more of the disclosed antibodies or antigen binding fragmentsthereof to reduce tumor burden in the subject.

One embodiment provides a method for treating an infection in a subjectin need thereof, by administering to the subject an effective amount ofone or more of the disclosed antibodies or antigen binding fragmentsthereof to treat the infection in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the interaction kinetics as a function of timebetween monoclonal antibody 4G9 and human PD-1. The graph shows tracesfrom concentrations of human PD-1 at 0, 125, 250, 500, 500, and 1000 nM.

FIG. 2 is a graph showing the interaction kinetics as a function of timebetween monoclonal antibody 4G9 and mouse PD-1. The graph shows tracesfrom concentrations of mouse PD-1 at 0, 62.5, 125, 500, 500, and 1000nM.

FIG. 3 is a graph showing the interaction kinetics as a function of timebetween monoclonal antibody 4C12 and human PD-1. The graph shows tracesfrom concentrations of human PD-1 at 0, 125, 250, 500, 500, and 1000 nM.

FIG. 4 is a graph showing the interaction kinetics as a function of timebetween monoclonal antibody 5C2 and human PD-1. The graph shows tracesfrom concentrations of mouse PD-1 at 0 and 1000 nM.

FIG. 5 is a graph showing the interaction kinetics as a function of timebetween monoclonal antibody 5C2 and mouse PD-1. The graph shows tracesfrom concentrations of human PD-1 at 0, 62.5, 125, 250, 500, 500, and1000 nM.

FIG. 6A is a flow cytometry histogram of EL4 cells stained with anisotype control antibody or commercial anti-PD-1 antibody J43. FIG. 6Bis a flow cytometry histogram of EL4 cells stained with secondaryantibody only or antibodies 4G9, 5C2, and 4C12. FIG. 6C is a flowcytometry histogram of E14 cells stained with secondary antibody only orantibody 4G9.

FIG. 6D is a flow cytometry histogram of E14 cells stained withsecondary antibody only or antibody 5C2. FIG. 6E is a flow cytometryhistogram of E14 cells stained with secondary antibody only or antibody4C12. FIG. 6F is a bar graph showing binding of various purified mousePD-1 antibodies to EL4 cells.

FIGS. 7A and 7B are bar graphs showing concentration of IFNγ (FIG. 7A)or IL-2 in supernatant from CD4 T cells treated with various antibodies.The X axis is treatment group and the Y axis is concentration (ng/ml).FIG. 7C is a bar graph showing IFNγ concentration in supernatant fromhuman CD4 T cells treated with 4G9 or 5C2 antibodies. The X axisrepresents treatment group and the Y axis represents concentration(ng/mL).

FIG. 8 is a bar graph showing levels of intracellular staining of pAKTin mouse CD4 T cells treated with various antibodies. The X axisrepresents treatment group and the Y axis represents pAKT (S473) MFI.

FIG. 9 is a Western blot showing IgG heavy chain and light chain in thevarious antibodies.

FIG. 10 is a bar graph showing binding of 4G9 and 5C2 antibodies tohuman PD-1-Fc. The X axis represents antibody concentration and the Yaxis represents OD450.

FIGS. 11A and 11B are flow cytometry histograms showing binding of 4G9and 5C2 antibodies to PD-1 in CD4 T cells from PD-1 KO mice (FIG. 11A)or PD-1 WT mice (FIG. 11B).

FIG. 12 is a line graph showing pS6 expression in CD4 T cells treatedwith 4G9 (

), 5C2 (

), commercial Ab-1 (

), commercial Ab-2 (

), or untreated (

). The axis represents concentration of total protein (μg/ml) and the Yaxis represents OD450,

FIG. 13A is a schematic illustration showing the experimental design forthe TC-1 tumor experiments. FIG. 13B is a line graph showing averagetumor volume (cm³) over time (days) for TC-1 tumor bearing mice treatedwith E7 Vax (□), 4G9 (Δ), RMP 1-14 (

), E7 Vax+RMP 1-14 (●), E7 Vax+4G9 (▴), or untreated (∘). The X axisrepresents time (days) and the Y axis represents average tumor volume(cm³). FIG. 13C is a line graph showing percent survival over time forTC-1 tumor bearing mice treated with E7 Vax (∘), 4G9 (●), RMP 1-14 (

), E7 Vax+RMP 1-14 (

), E7 Vax+4G9 (▴), or untreated (Δ). FIG. 13D is a line graph showingaverage tumor volume (cm³) over time (days) for TC-1 tumor bearing micetreated with E7 Vax (▪), 4C12 (▾), 5C2 (♦), RMP 1-14 (●), E7 Vax+RMP1-14 (

), E7 Vax÷4C12 (

), E7 Vax+5C2 (

), or untreated (∘).

FIG. 14A is a schematic illustration of the experimental design for theTC-1 tumor experiments. FIG. 14B is a line graph showing average tumorvolume (cm³) over time (days) for TC-1 tumor bearing mice treated withE7 Vax (□), 4G9 (Δ), RMP 1-14 (

), J43 (

), E7 Vax+4G9 (

), E7 Vax+RMP 1-14 (

), E7 Vax+J43 (●), or untreated (

). The X axis represents time (days) and the Y axis represents averagetumor volume (cm³). FIG. 14C is a line graph showing percent survivalover time of TC-1 tumor bearing mice treated with E7 Vax+(□), 4G9 (Δ),4C12 (∇), 5C2 (⋄), RMP 1-14 (

), J43 (

), E7 Vax+4G9 (

), E7 Vax+4C12 (

), E7 Vax+5C2 (

), E7 Vax+RMP 1-14 (

), E7 Vax+J43 (

), or untreated (

). The X axis represents time (days) and the Y axis represents percentsurvival.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

As used herein, a molecule is said to be able to “immunospecificallybind” a second molecule if such binding exhibits the specificity andaffinity of an antibody to its cognate antigen. Antibodies are said tobe capable of immunospecifically binding to a target region orconformation (“epitope”) of an antigen if such binding involves theantigen recognition site of the immunoglobulin molecule. An antibodythat immunospecifically binds to a particular antigen may bind to otherantigens with lower affinity if the other antigen has some sequence orconformational similarity that is recognized by the antigen recognitionsite as determined by, e.g., immunoassays, BIACORE® assays, or otherassays known in the art, but would not bind to a totally unrelatedantigen. Preferably, however, antibodies (and their antigen bindingfragments) will not cross-react with other antigens. Antibodies may alsobind to other molecules in a way that is not immunospecific, such as toFcR receptors, by virtue of binding domains in other regions/domains ofthe molecule that do not involve the antigen recognition site, such asthe Fc region.

As used herein, a molecule is said to “physiospecifically bind” a secondmolecule if such binding exhibits the specificity and affinity of areceptor to its cognate binding ligand. A molecule can be capable ofphysiospecifically binding to more than one other molecule.

As used herein, the term “antibody” is intended to denote animmunoglobulin molecule that possesses a “variable region” antigenrecognition site. The term “variable region” is intended to distinguishsuch domain of the immunoglobulin from domains that are broadly sharedby antibodies (such as an antibody Fc domain). The variable regionincludes a “hypervariable region” whose residues are responsible forantigen binding. The hypervariable region includes amino acid residuesfrom a “Complementarity Determining Region” or “CDR” (i.e., typically atapproximately residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in thelight chain variable domain and at approximately residues 27-35 (H1),50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991))and/or those residues from a “hypervariable loop” (i.e., residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). “FrameworkRegion” or “FR” residues are those variable domain residues other thanthe hypervariable region residues as herein defined. The term antibodyincludes monoclonal antibodies, multi-specific antibodies, humanantibodies, humanized antibodies, synthetic antibodies, chimericantibodies, camelized antibodies (See e.g., Muyldermans et al., 2001,Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech.1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231:25;International Publication Nos. WO 94/04678 and WO 94/25591; U.S. Pat.No. 6,005,079), single-chain Fvs (scFv) (see, e.g., see Pluckthun in ThePharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds. Springer-Verlag, New York, pp. 269-315 (1994)), single chainantibodies, disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic(anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Idantibodies to antibodies). In particular, such antibodies includeimmunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA andIgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass.

As used herein, the term “antigen binding fragment” of an antibodyrefers to one or more portions of an antibody that contain theantibody's Complementarity Determining Regions (“CDRs”) and optionallythe framework residues that include the antibody's “variable region”antigen recognition site, and exhibit an ability to immunospecificallybind antigen. Such fragments include Fab′, F(ab′)₂, Fv, single chain(ScFv), and mutants thereof, naturally occurring variants, and fusionproteins including the antibody's “variable region” antigen recognitionsite and a heterologous protein (e.g., a toxin, an antigen recognitionsite for a different antigen, an enzyme, a receptor or receptor ligand,etc.).

As used herein, the term “fragment” refers to a peptide or polypeptideincluding an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least 80 contiguous amino acid residues, atleast 90 contiguous amino acid residues, at least 100 contiguous aminoacid residues, at least 125 contiguous amino acid residues, at least 150contiguous amino acid residues, at least 175 contiguous amino acidresidues, at least 200 contiguous amino acid residues, or at least 250contiguous amino acid residues.

The term “binding molecule,” as used herein is intended to refer tomolecules that specifically interact with and bind to a particulartarget. The target can comprise a biologic or small (chemical) molecule.The target molecule may define an antigen or antigenic moiety. Examplesof a binding molecule include, but are not limited to, antibodies(including monoclonal antibodies, bispecific antibodies, as well asantibody fragments), fusion proteins, and other antigen-binding moleculeknown to those skilled in the art.

As used herein the term “modulate” relates to a capacity to alter aneffect, result, or activity (e.g., signal transduction). Such modulationcan agonistic or antagonistic. Antagonistic modulation can be partial(i.e., attenuating, but not abolishing) or it can completely abolishsuch activity (e.g., neutralizing). Modulation can includeinternalization of a receptor following binding of an antibody or areduction in expression of a receptor on the target cell. Agonisticmodulation can enhance or otherwise increase or enhance an activity(e.g., signal transduction). In a still further embodiment, suchmodulation can alter the nature of the interaction between a ligand andits cognate receptor so as to alter the nature of the elicited signaltransduction. For example, the molecules can, by binding to the ligandor receptor, alter the ability of such molecules to bind to otherligands or receptors and thereby alter their overall activity.Preferably, such modulation will provide at least a 10% change in ameasurable immune system activity, more preferably, at least a 50%change in such activity, or at least a 2-fold, 5-fold, 10-fold, or stillmore preferably, at least a 100-fold change in such activity.

The term “substantially,” as used in the context of binding or exhibitedeffect, is intended to denote that the observed effect isphysiologically or therapeutically relevant. Thus, for example, amolecule is able to substantially block an activity of a ligand orreceptor if the extent of blockage is physiologically or therapeuticallyrelevant (for example if such extent is greater than 60% complete,greater than 70% complete, greater than 75% complete, greater than 80%complete, greater than 85% complete, greater than 90% complete, greaterthan 95% complete, or greater than 97% complete). Similarly, a moleculeis said to have substantially the same immunospecificity and/orcharacteristic as another molecule, if such immunospecificities andcharacteristics are greater than 60% identical, greater than 70%identical, greater than 75% identical, greater than 80% identical,greater than 85% identical, greater than 90% identical, greater than 95%identical, or greater than 97% identical).

As used herein, the “co-stimulatory” signals encompass positiveco-stimulatory signals (e.g., signals that result in enhancing anactivity) and negative co-stimulatory signals (e.g., signals that resultin inhibiting an activity).

As used herein, the term “derivative” refers to an antibody orantigen-binding fragment thereof that immunospecifically binds to thesame target of a parent or reference antibody but which differs in aminoacid sequence from the parent or reference antibody or antigen bindingfragment thereof by including one, two, three, four, five or more aminoacid substitutions, additions, deletions or modifications relative tothe parent or reference antibody or antigen binding fragment thereof.Preferably such derivatives will have substantially the sameimmunospecificity and/or characteristics, or the same immunospecificityand characteristics as the parent or reference antibody or antigenbinding fragment thereof. The amino acid substitutions or additions ofsuch derivatives can include naturally occurring (i.e., DNA-encoded) ornon-naturally occurring amino acid residues. The term “derivative”encompasses, for example, chimeric or humanized variants, as well asvariants having altered CH1, hinge, CH2, CH3 or CH4 regions, so as toform, for example antibodies, etc., having variant Fc regions thatexhibit enhanced or impaired effector or binding characteristics.

As used herein, a “chimeric antibody” is a molecule in which differentportions of the antibody are derived from different immunoglobulinmolecules such as antibodies having a variable region derived from anon-human antibody and a human immunoglobulin constant region.

As used herein, the term “humanized antibody” refers to animmunoglobulin including a human framework region and one or more CDR'sfrom a non-human (usually a mouse or rat) immunoglobulin. The non-humanimmunoglobulin providing the CDR's is called the “donor” and the humanimmunoglobulin providing the framework is called the “acceptor.”Constant regions need not be present, but if they are, they should besubstantially identical to human immunoglobulin constant regions, i.e.,at least about 85-99%, preferably about 95% or more identical. Hence,all parts of a humanized immunoglobulin, except possibly the CDR's, aresubstantially identical to corresponding parts of natural humanimmunoglobulin sequences. A humanized antibody is an antibody includinga humanized light chain and a humanized heavy chain immunoglobulin. Forexample, a humanized antibody would not encompass a typical chimericantibody, because, e.g., the entire variable region of a chimericantibody is non-human.

As used herein, the term “endogenous concentration” refers to the levelat which a molecule is natively expressed (i.e., in the absence ofexpression vectors or recombinant promoters) by a cell (which cell canbe a normal cell, a cancer cell or an infected cell).

As used herein, the terms “treat,” “treating,” “treatment” and“therapeutic use” refer to the elimination, reduction or amelioration ofone or more symptoms of a disease or disorder exacerbated by anti-PD-1antibodies or an antigen fragment thereof.

As used herein, a “therapeutically effective amount” refers to thatamount of a therapeutic agent sufficient to mediate a clinicallyrelevant elimination, reduction or amelioration of such symptoms. Aneffect is clinically relevant if its magnitude is sufficient to impactthe health or prognosis of a recipient subject. A therapeuticallyeffective amount may refer to the amount of therapeutic agent sufficientto delay or minimize the onset of disease, e.g., delay or minimize thespread of cancer. A therapeutically effective amount may also refer tothe amount of the therapeutic agent that provides a therapeutic benefitin the treatment or management of a disease.

As used herein, the term “prophylactic agent” refers to an agent thatcan be used in the prevention of a disorder or disease prior to thedetection of any symptoms of such disorder or disease. A“prophylactically effective” amount is the amount of prophylactic agentsufficient to mediate such protection. A prophylactically effectiveamount may also refer to the amount of the prophylactic agent thatprovides a prophylactic benefit in the prevention of disease.

As used herein, the term “cancer” refers to a neoplasm or tumorresulting from abnormal uncontrolled growth of cells. As used herein,cancer explicitly includes, leukemias and lymphomas. The term “cancer”refers to a disease involving cells that have the potential tometastasize to distal sites and exhibit phenotypic traits that differfrom those of non-cancer cells, for example, formation of colonies in athree-dimensional substrate such as soft agar or the formation oftubular networks or web-like matrices in a three-dimensional basementmembrane or extracellular matrix preparation. Non-cancer cells do notform colonies in soft agar and form distinct sphere-like structures inthree-dimensional basement membrane or extracellular matrixpreparations.

As used herein, an “immune cell” refers to any cell from the hemopoieticorigin including, but not limited to, T cells, B cells, monocytes,dendritic cells, and macrophages.

As used herein, “valency” refers to the number of binding sitesavailable per molecule.

As used herein, the terms “immunologic,” “immunological” or “immune”response is the development of a beneficial humoral (antibody mediated)and/or a cellular (mediated by antigen-specific T cells or theirsecretion products) response directed against a peptide in a recipientpatient. Such a response can be an active response induced byadministration of immunogen or a passive response induced byadministration of antibody or primed T-cells. A cellular immune responseis elicited by the presentation of polypeptide epitopes in associationwith Class I or Class II WIC molecules to activate antigen-specific CD4⁺T helper cells and/or CD8⁺ cytotoxic T cells. The response may alsoinvolve activation of monocytes, macrophages, NK cells, basophils,dendritic cells, astrocytes, microglia cells, eosinophils, activation orrecruitment of neutrophils or other components of innate immunity. Thepresence of a cell-mediated immunological response can be determined byproliferation assays (CD4⁺ T cells) or CTL (cytotoxic T lymphocyte)assays. The relative contributions of humoral and cellular responses tothe protective or therapeutic effect of an immunogen can bedistinguished by separately isolating antibodies and T-cells from animmunized syngeneic animal and measuring protective or therapeuticeffect in a second subject.

As used herein, an “immunogenic agent” or “immunogen” is capable ofinducing an immunological response against itself on administration to amammal, optionally in conjunction with an adjuvant.

As used herein, the terms “individual,” “host,” “subject, and “patient”are used interchangeably herein, and refer to a mammal, including, butnot limited to, humans, rodents, such as mice and rats, and otherlaboratory animals.

As used herein, the term “polypeptide” refers to a chain of amino acidsof any length, regardless of modification (e.g., phosphorylation orglycosylation). The term polypeptide includes proteins and fragmentsthereof. The polypeptides can be “exogenous,” meaning that they are“heterologous,” i.e., foreign to the host cell being utilized, such ashuman polypeptide produced by a bacterial cell. Polypeptides aredisclosed herein as amino acid residue sequences. Those sequences arewritten left to right in the direction from the amino to the carboxyterminus. In accordance with standard nomenclature, amino acid residuesequences are denominated by either a three letter or a single lettercode as indicated as follows: Alanine (Ala, A), Arginine (Arg, R),Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C),Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine(His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K),Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine(Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y),and Valine (Val, V).

As used herein, the term “variant” refers to a polypeptide orpolynucleotide that differs from a reference polypeptide orpolynucleotide, but retains essential properties. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more modifications (e.g.,substitutions, additions, and/or deletions). A substituted or insertedamino acid residue may or may not be one encoded by the genetic code. Avariant of a polypeptide may be naturally occurring such as an allelicvariant, or it may be a variant that is not known to occur naturally.

Modifications and changes can be made in the structure of thepolypeptides of the disclosure and still obtain a molecule havingsimilar characteristics as the polypeptide (e.g., a conservative aminoacid substitution). For example, certain amino acids can be substitutedfor other amino acids in a sequence without appreciable loss ofactivity. Because it is the interactive capacity and nature of apolypeptide that defines that polypeptide's biological functionalactivity, certain amino acid sequence substitutions can be made in apolypeptide sequence and nevertheless obtain a polypeptide with likeproperties.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a polypeptide is generallyunderstood in the art. It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still result in a polypeptide with similar biologicalactivity. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. Those indicesare: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8);glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9);tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5);glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9);and arginine (−4.5).

It is believed that the relative hydropathic character of the amino aciddetermines the secondary structure of the resultant polypeptide, whichin turn defines the interaction of the polypeptide with other molecules,such as enzymes, substrates, receptors, antibodies, antigens, andcofactors. It is known in the art that an amino acid can be substitutedby another amino acid having a similar hydropathic index and stillobtain a functionally equivalent polypeptide. In such changes, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis ofhydrophilicity, particularly where the biological functional equivalentpolypeptide or peptide thereby created is intended for use inimmunological embodiments. The following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); proline (−0.5±1); threonine(−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood thatan amino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent, and inparticular, an immunologically equivalent polypeptide. In such changes,the substitution of amino acids whose hydrophilicity values are within±2 is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various foregoingcharacteristics into consideration are well known to those of skill inthe art and include (original residue: exemplary substitution): (Ala:Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln:Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu:Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip:Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Embodiments of thisdisclosure thus contemplate functional or biological equivalents of apolypeptide as set forth above. In particular, embodiments of thepolypeptides can include variants having about 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99%, or more sequence identity to the polypeptide ofinterest.

The term “percent (%) sequence identity” is defined as the percentage ofnucleotides or amino acids in a candidate sequence that are identicalwith the nucleotides or amino acids in a reference nucleic acidsequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN,ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full-length of the sequences being compared can bedetermined by known methods.

For purposes herein, the % sequence identity of a given nucleotides oramino acids sequence C to, with, or against a given nucleic acidsequence D (which can alternatively be phrased as a given sequence Cthat has or comprises a certain % sequence identity to, with, or againsta given sequence D) is calculated as follows:

100 times the fraction W/Z, where W is the number of nucleotides oramino acids scored as identical matches by the sequence alignmentprogram in that program's alignment of C and D, and where Z is the totalnumber of nucleotides or amino acids in D. It will be appreciated thatwhere the length of sequence C is not equal to the length of sequence D,the % sequence identity of C to D will not equal the % sequence identityof D to C.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water and emulsions such as anoil/water or water/oil emulsion, and various types of wetting agents.

As used herein, the terms “antigenic determinant” and “epitope” are usedinterchangeably and refer to the structure recognized by an antibody.

As used herein, a “conformational epitope” is an epitope that includesdiscontinuous sections of the antigen's amino acid sequence. Antibodiesbind a conformational epitope based on 3-D surface features, shape, ortertiary structure of the antigen.

As used herein, a “linear epitope” is an epitope that formed by acontinuous sequence of amino acids from the antigen. Linear epitopestypically include about 5 to about 10 continuous amino acid residues.Antibodies bind a linear epitope based on the primary sequence of theantigen.

As used herein, a “paratope,” also called an “antigen-binding site,” isa part of an antibody which recognizes and binds to an antigen.

II. Compositions

Antibodies and antigen binding fragments thereof that immunospecificallybind to PD-1 are provided. Contrary to the existing paradigm that PD-1exclusively promotes a suppressive immune response (Riley, J., ImmunolRev. 229(1):114-125 (2009)), the disclosed antibodies and antigenbinding fragments thereof, immunospecifically bind to PD-1 and cause anactivating signal to be delivered to the immune cell that activates theimmune cell rather than suppressing the immune cell.

A. Programmed Death Receptor Protein 1 (PD-1)

The disclosed antibodies and antigen binding fragments thereofimmunospecifically bind to PD-1. The antibodies and antigen bindingfragments thereof can bind to PD-1 having for example the amino acidsequences provide below.

Amino acid sequences for human PD-1 and mouse PD-1 are known in the artand include, for example,

human PD-1 (SEQ ID NO: 1)MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLAccession: AJS10360 and which is specificallyincorporated by reference in its entirety. mouse PD-1 (SEQ ID NO: 2)MWVRQVPWSFTWAVLQLSWQSGWLLEVPNGPWRSLTFYPAWLTVSEGANATFTCSLSNWSEDLMLNWNRLSPSNQTEKQAAFCNGLSQPVQDARFQIIQLPNRHDFHMNILDTRRNDSGIYLCGAISLHPKAKIEESPGAELVVTERILETSTRYPSPSPKPEGRFQGMVIGIIVISALVGIPVLLLLAWALAVFCSTSMSEARGAGSKDDTLKEEPSAAPVPSVAYEELDFQGREKTPELPTACVHTEYATIVFTEGLGASAMGRRGSADGLQGPRPPRHEDGHCSWPLUniProtKB-Q02242 (PDCD1_MOUSE) and which isspecifically incorporated by reference in its entirety.

B. Antibody Compositions

The disclosed anti-PD-1 antibodies or antigen binding fragments thereofinclude whole immunoglobulin (i.e., an intact antibody) of any class,fragments thereof, and synthetic proteins containing at least theantigen binding variable domain of an antibody. In some embodiments, thedisclosed antibody contains both an antibody light chain as well as atleast the variable domain of an antibody heavy chain. In otherembodiments, such molecules can further include one or more of the CH1,hinge, CH2, CH3, and CH4 regions of the heavy chain (especially, the CH1and hinge regions, or the CH1, hinge and CH2 regions, or the CH1, hinge,CH2 and CH3 regions). The antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including IgG₁, IgG₂, IgG₃ and IgG₄. In some embodiments, the constantdomain is a complement fixing constant domain where it is desired thatthe antibody exhibit cytotoxic activity, and the class is typicallyIgG₁. In other embodiments, where such cytotoxic activity is notdesirable, the constant domain can be of the IgG₂ or IgG₄ class. Theantibody can include sequences from more than one class or isotype, andselecting particular constant domains to optimize desired effectorfunctions is within the ordinary skill in the art.

The variable domains differ in sequence among antibodies and are used inthe binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not usually evenlydistributed through the variable domains of antibodies. It is typicallyconcentrated in three segments called complementarity determiningregions (CDRs) or hypervariable regions both in the light chain and theheavy chain variable domains. The more highly conserved portions of thevariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a beta-sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the beta-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen binding site of antibodies.

Some embodiments provide fragments of the anti-PD-1 antibodies whichhave bioactivity. The fragments, whether attached to other sequences ornot, may include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the nonmodified antibody or antibody fragment.

Another embodiment provides single-chain antibodies specific to PD-1.Methods for the production of single-chain antibodies are well known tothose of skill in the art. A single chain antibody can be created byfusing together the variable domains of the heavy and light chains usinga short peptide linker, thereby reconstituting an antigen binding siteon a single molecule. Single-chain antibody variable fragments (scFvs)in which the C-terminus of one variable domain is tethered to theN-terminus of the other variable domain via a 15 to 25 amino acidpeptide or linker have been developed without significantly disruptingantigen binding or specificity of the binding. The linker is chosen topermit the heavy chain and light chain to bind together in their properconformational orientation.

Another embodiment provides divalent single-chain variable fragments(di-scFvs) that can be engineered by linking two scFvs. This can be doneby producing a single peptide chain with two VH and two VL regions,yielding tandem scFvs. ScFvs can also be designed with linker peptidesthat are too short for the two variable regions to fold together (aboutfive amino acids), forcing scFvs to dimerize. This type is known asdiabodies. Diabodies have been shown to have dissociation constants upto 40-fold lower than corresponding scFvs, meaning that they have a muchhigher affinity to their target. Still shorter linkers (one or two aminoacids) lead to the formation of trimers (triabodies or tribodies).Tetrabodies have also been produced. They exhibit an even higheraffinity to their targets than diabodies.

Another embodiment provides a monoclonal antibody specific to PD-1 thatinduces an activating signal to immune cells. The monoclonal antibodycan be obtained from a substantially homogeneous population ofantibodies, i.e., the individual antibodies within the population areidentical except for possible naturally occurring mutations that may bepresent in a small subset of the antibody molecules. Monoclonalantibodies include “chimeric” antibodies in which a portion of the heavyand/or light chain is identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, as long asthey exhibit the desired antagonistic activity.

1. Chimeric and Humanized Antibodies

Another embodiment provides chimeric anti-PD-1 antibodies and antigenbinding fragments thereof including one or more of the disclosedsequences and functional variants thereof are also provided that bind toPD-1 and cause an activating signal to be transmitted in to an immunecell expressing PD-1.

Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S.Pat. Nos. 6,311,415, 5,807,715, 4,816,567, and 4,816,397. Chimericantibodies including one or more CDRs from a non-human species andframework regions from a human immunoglobulin molecule can be producedusing a variety of techniques known in the art including, for example,CDR-grafting (EP 239,400; International Publication No. WO 91/09967; andU.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering orresurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology28(4/5):489-498; Studnicka et al., 1994, Protein Engineering 7:805; andRoguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969), and chainshuffling (U.S. Pat. No. 5,565,332).

The disclosed anti-PD-1 antibodies or antigen binding fragments thereofcan be human or humanized antibodies, or antigen binding fragmentsthereof. Many non-human antibodies (e.g., those derived from mice, rats,or rabbits) are naturally antigenic in humans, and thus can give rise toundesirable immune responses when administered to humans. Therefore, theuse of human or humanized antibodies in the methods serves to lessen thechance that an antibody administered to a human will evoke anundesirable immune response.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region (J(H)) gene in chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production.Transfer of the human germ-line immunoglobulin gene array in suchgerm-line mutant mice will result in the production of human antibodiesupon antigen challenge.

Optionally, the antibodies are generated in other species and“humanized” for administration in humans. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂, or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementarity determining region (CDR) of the recipientantibody are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also contain residues thatare found neither in the recipient antibody nor in the imported CDR orframework sequences. In general, the humanized antibody will containsubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will contain at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art,see, for example, European Patent Nos. EP 239,400, EP 592,106, and EP519,596; International Publication Nos. WO 91/09967 and WO 93/17105;U.S. Pat. Nos. 5,225,539, 5,530,101, 5,565,332, 5,585,089, 5,766,886,and 6,407,213; and Padlan, 1991, Molecular Immunology 28(4/5):489-498;Studnicka et al., 1994, Protein Engineering 7(6):805-814; Roguska etal., 1994, PNAS 91:969-973; Tan et al., 2002, J. Immunol. 169:1119-1125;Caldas et al., 2000, Protein Eng. 13:353-360; Morea et al., 2000,Methods 20:267-79; Baca et al., 1997, J. Biol. Chem. 272:10678-10684;Roguska et al., 1996, Protein Eng. 9:895-904; Couto et al., 1995, CancerRes. 55 (23 Supp):5973s-5977s; Couto et al., 1995, Cancer Res.55:1717-22; Sandhu, 1994, Gene 150:409-10; Pedersen et al., 1994, J.Mol. Biol. 235:959-973; Jones et al., 1986, Nature 321:522-525;Reichmann et al., 1988, Nature 332:323-329; and Presta, 1992, Curr. Op.Struct. Biol. 2:593-596).

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Antibodyhumanization techniques generally involve the use of recombinant DNAtechnology to manipulate the DNA sequence encoding one or morepolypeptide chains of an antibody molecule. Humanization can beessentially performed by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Accordingly, ahumanized form of a non-human antibody (or a fragment thereof) is achimeric antibody or fragment, wherein substantially less than an intacthuman variable domain has been substituted by the corresponding sequencefrom a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues and possibly someFR residues are substituted by residues from analogous sites in rodentantibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies can be very important in order toreduce antigenicity. According to the “best-fit” method, the sequence ofthe variable domain of a rodent antibody is screened against the entirelibrary of known human variable domain sequences. The human sequencewhich is closest to that of the rodent is then accepted as the humanframework (FR) for the humanized antibody. Another method uses aparticular framework derived from the consensus sequence of all humanantibodies of a particular subgroup of light or heavy chains. The sameframework may be used for several different humanized antibodies.

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, humanized antibodies can be prepared by a processof analysis of the parental sequences and various conceptual humanizedproducts using three dimensional models of the parental and humanizedsequences. Three dimensional immunoglobulin models are commonlyavailable and are familiar to those skilled in the art. Computerprograms are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the consensus andimport sequence so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the CDR residues are directly and most substantially involved ininfluencing antigen binding.

A human, humanized or chimeric antibody derivative can includesubstantially all of at least one, and typically two, variable domainsin which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin (i.e., donor antibody) and all orsubstantially all of the framework regions are those of a humanimmunoglobulin consensus sequence. Such antibodies can also include atleast a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin. The constant domains of such antibodiescan be selected with respect to the proposed function of the antibody,in particular the effector function which may be required. In someembodiments, the constant domains of such antibodies are or can includehuman IgA, IgD, IgE, IgG or IgM domains. In a specific embodiment, humanIgG constant domains, especially of the IgG1 and IgG3 isotypes are used,when the humanized antibody derivative is intended for a therapeutic useand antibody effector functions such as antibody-dependent cell-mediatedcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activityare needed. In alternative embodiments, IgG2 and IgG4 isotypes are usedwhen the antibody is intended for therapeutic purposes and antibodyeffector function is not required. Fc constant domains including one ormore amino acid modifications which alter antibody effector functionssuch as those disclosed in U.S. Patent Application Publication Nos.2005/0037000 and 2005/0064514.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor CDR orthe consensus framework can be mutagenized by substitution, insertion ordeletion of at least one residue so that the CDR or framework residue atthat site does not correspond to either the consensus or the donorantibody. In some embodiments, such mutations are not extensive.Usually, at least 75% of the humanized antibody residues will correspondto those of the parental framework region (FR) and CDR sequences, moreoften 90%, or greater than 95%. Humanized antibodies can be producedusing variety of techniques known in the art, including, but not limitedto, CDR-grafting (European Patent No. EP 239,400; InternationalPublication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101,and 5,585,089), veneering or resurfacing (European Patent Nos. EP592,106 and EP 519,596; Padlan, 1991, Molecular Immunology28(4/5):489-498; Studnicka et al., 1994, Protein Engineering7(6):805-814; and Roguska et al., 1994, Proc. Natl. Acad. Sci.91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and techniquesdisclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886, 5,585,089,International Publication No. WO 9317105, Tan et al., 2002, J. Immunol.169:1119-25, Caldas et al., 2000, Protein Eng. 13:353-60, Morea et al.,2000, Methods 20:267-79, Baca et al., 1997, J. Biol. Chem. 272:10678-84,Roguska et al., 1996, Protein Eng. 9:895-904, Couto et al., 1995, CancerRes. 55 (23 Supp):5973s-5977s, Couto et al., 1995, Cancer Res.55:1717-22, Sandhu, 1994, Gene 150:409-10, Pedersen et al., 1994, J.Mol. Biol. 235:959-73, Jones et al., 1986, Nature 321:522-525, Riechmannet al., 1988, Nature 332:323, and Presta, 1992, Curr. Op. Struct. Biol.2:593-596.

Often, framework residues in the framework regions will be substitutedwith the corresponding residue from the CDR donor antibody to alter, forexample improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; U.S. Publication Nos.2004/0049014 and 2003/0229208; U.S. Pat. Nos. 6,350,861; 6,180,370;5,693,762; 5,693,761; 5,585,089; and 5,530,101 and Riechmann et al.,1988, Nature 332:323).

Human, chimeric or humanized derivatives of the disclosed murineanti-human Siglec-15 antibodies can be used for in vivo methods inhumans. Murine antibodies or antibodies of other species can beadvantageously employed for many uses (for example, in vitro or in situdetection assays, acute in vivo use, etc.). Such a human or humanizedantibody can include amino acid residue substitutions, deletions oradditions in one or more non-human CDRs. The humanized antibodyderivative can have substantially the same binding, stronger binding orweaker binding when compared to a non-derivative humanized antibody. Inspecific embodiments, one, two, three, four, or five amino acid residuesof the CDR have been substituted, deleted or added (i.e., mutated).Completely human antibodies are particularly desirable for therapeutictreatment of human subjects.

Such human antibodies can be made by a variety of methods known in theart including phage display methods using antibody libraries derivedfrom human immunoglobulin sequences (see U.S. Pat. Nos. 4,444,887 and4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433,WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741).Such human antibodies can be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes.

For example, the human heavy and light chain immunoglobulin genecomplexes can be introduced randomly or by homologous recombination intomouse embryonic stem cells. Alternatively, the human variable region,constant region, and diversity region can be introduced into mouseembryonic stem cells in addition to the human heavy and light chaingenes. The mouse heavy and light chain immunoglobulin genes can berendered non-functional separately or simultaneously with theintroduction of human immunoglobulin loci by homologous recombination.In particular, homozygous deletion of the JH region prevents endogenousantibody production. The modified embryonic stem cells are expanded andmicroinjected into blastocysts to produce chimeric mice. The chimericmice are then bred to produce homozygous offspring which express humanantibodies. The transgenic mice are immunized using conventionalmethodologies with a selected antigen, e.g., all or a portion of apolypeptide. Monoclonal antibodies directed against the antigen can beobtained from the immunized, transgenic mice using conventionalhybridoma technology (see, e.g., U.S. Pat. No. 5,916,771).

The human immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview of this technology for producing humanantibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93,which is incorporated herein by reference in its entirety). For adetailed discussion of this technology for producing human antibodiesand human monoclonal antibodies and protocols for producing suchantibodies, see, e.g., International Publication Nos. WO 98/24893, WO96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126,5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598,which are incorporated by reference herein in their entirety. Inaddition, companies can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

DNA sequences coding for human acceptor framework sequences include butare not limited to FR segments from the human germline VH segment VH1-18and JH6 and the human germline VL segment VK-A26 and JK4. In a specificembodiment, one or more of the CDRs are inserted within frameworkregions using routine recombinant DNA techniques. The framework regionscan be naturally occurring or consensus framework regions, and humanframework regions (see, e.g., Chothia et al., 1998, “StructuralDeterminants In The Sequences Of Immunoglobulin Variable Domain,” J.Mol. Biol. 278: 457-479 for a listing of human framework regions).

C. Antibody Sequences

1. 4C12 Heavy Chain Sequences

One embodiment provides a murine monoclonal antibody or antigen bindingfragment thereof isolated from hybridoma 4C12.

Another embodiment provides an antibody or antigen binding fragmentthereof having a heavy chain encoded by a nucleic acid with at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to:

(SEQ ID NO: 3)

TGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGGTGTCCTGCAAGGCTTCTGG

CTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGT

Underlined sequences correspond to complementarity determining regions(CDRs). Double underline sequence corresponds to the constant region.Dashed underlined sequences correspond to the leader sequence.

The nucleic acid can be in a vector, for example an expression vector.The nucleic acid can be extrachromosal on inserted into a chromosome ofa host cell, for example a Chinese Hamster Ovary cell.

One embodiment provides an antibody or antigen binding fragment thereofhaving heavy chain with an amino acid sequence having at least 50%, 60%,70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to:

(SEQ ID NO: 4)

Single underline corresponds to the leader sequence. Double under linecorresponds to CDRs, and dashed underline corresponds to the constantregion.

Another embodiment provides an antibody or antigen binding fragmentthereof having heavy chain without the leader sequence with an aminoacid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or100% sequence identity to:

(SEQ ID NO: 5)

Double under line corresponds to CDRs, and broken underline correspondsto the constant region.

The amino acid sequence for CDR1 of the 4C12 heavy chain is

(SEQ ID NO: 6) SYWMH.

The amino acid sequence for CDR2 of the 4C12 heavy chain is

(SEQ ID NO: 7) RIHPSDSDTNYNQKFKG.

The amino acid sequence for CDR3 of the 4C12 heavy chain is

(SEQ ID NO: 8) YGNYASGFAY.

One embodiment provides an antibody or antigen binding fragment thereofhaving a heavy chain according to SEQ ID NO:4 or 5.

Another embodiment provides and antibody or antigen binding fragmentthereof having a heavy chain encoded by a nucleic acid with at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQID NO:3.

One embodiment provides an antibody having three different CDRs selectedfrom the group consisting of SEQ ID NO: 6, 7, and 8.

2. 4C12 Light Chain Sequences

Another embodiment provides an antibody or antigen binding fragmentthereof having a light chain encoded by a nucleic acid with at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to:

(SEQ ID NO: 9)

CACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCAACAAAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTATACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCACTTTATTACTG

Dashed underline represents the leader sequence. Single underlinerepresents the CDRs. Double underline represents the constant region.

The nucleic acid can be in a vector, for example an expression vector.The nucleic acid can be extrachromosal on inserted into a chromosome ofa host cell, for example a Chinese Hamster Ovary cell.

Another embodiment provides an antibody or antigen binding fragmentthereof having a light chain having an amino acid sequence with at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to:

(SEQ ID NO: 10)

Underline represents the leader sequence. Double underline representsCDRs. Dashed underline represents the constant region.

Another embodiment provides an antibody or antigen binding fragmentthereof having a light chain without the leader sequence having an aminoacid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or100% sequence identity to:

(SEQ ID NO: 11)

Double underline represents CDRs. Dashed underline represents theconstant region.

The amino acid sequence for CDR1 of the 4C12 light chain is

(SEQ ID NO: 12) KASQDVSTAVA.

The amino acid sequence for CDR2 of the 4C12 light chain is

(SEQ ID NO: 13) WASTRHT.

The amino acid sequence for CDR3 of the 4C12 light chain is

(SEQ ID NO: 14) QQHYSTPWT.

One embodiment provides an antibody or antigen binding fragment thereofhaving a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%,99%, or 100% sequence identity to SEQ ID NO:10 or 11.

Another embodiment provides and antibody or antigen binding fragmentthereof having a light chain encoded by a nucleic acid with at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQID NO:9.

One embodiment provides an antibody having three different CDRs selectedfrom the group consisting of SEQ ID NO: 12, 13, and 14.

Another embodiment provides an antibody or antigen binding fragmentthereof having a heavy chain with an amino acid sequence having at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQID NO:4 or 5 and light chain with an amino acid sequence at least 50%,60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ IDNO:10 or 11, or light and heavy chain combinations thereof.

Another embodiment provides an antibody or antigen binding fragmentthereof having three different heavy chain CDRs with an amino acidselected from the group consisting of SEQ ID NOs:6, 7, and 8 and threedifferent light chain CDRs with amino acids selected from the groupconsisting of SEQ ID NOs: 12, 13, and 14.

3. 2B5 Heavy Chain Sequences

One embodiment provides a murine monoclonal antibody or antigen bindingfragment thereof isolated from hybridoma 2B5.

Another embodiment provides an antibody or antigen binding fragmentthereof having a heavy chain encoded by a nucleic acid with at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to:

(SEQ ID NO: 15)

TGCAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACGCCTTCACTAATTACTTGATAGAGTGGGTAAAGCAGAGGCCTGGACAGGGCCTTGAGTGGATTGGAGTGATTAATCCTGGAAGTGGTGGTACTAACTACAATGAGAAGTTCAAGGGCAAGGCAACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCAGCTCAGGCCCCTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCC

Underlined sequences correspond to complementarity determining regions(CDRs). Double underline sequence corresponds to the constant region.Dashed underlined sequences correspond to the leader sequence.

The nucleic acid can be in a vector, for example an expression vector.The nucleic acid can be extrachromosal on inserted into a chromosome ofa host cell, for example a Chinese Hamster Ovary cell.

One embodiment provides an antibody or antigen binding fragment thereofhaving heavy chain with an amino acid sequence having at least 50%, 60%,70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to:

(SEQ ID NO: 16)

Single underline corresponds to the leader sequence. Double under linecorresponds to CDRs, and dashed underline corresponds to the constantregion.

Another embodiment provides an antibody or antigen binding fragmentthereof having heavy chain without the leader sequence with an aminoacid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or100% sequence identity to:

(SEQ ID NO: 17)

Double under line corresponds to CDRs, and dashed underline correspondsto the constant region.

The amino acid sequence for CDR1 of the 2B5 heavy chain is

(SEQ ID NO: 18) NYLIE.

The amino acid sequence for CDR2 of the 2B5 heavy chain is

(SEQ ID NO: 19) VINPGSGGTNYNEKFKG.

The amino acid sequence for CDR3 of the 2B5 heavy chain is

(SEQ ID NO: 20) SAQAPDY.

One embodiment provides an antibody or antigen binding fragment thereofhaving a heavy chain according to SEQ ID NO:16 or 17.

Another embodiment provides and antibody or antigen binding fragmentthereof having a heavy chain encoded by a nucleic acid with at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQID NO:15.

One embodiment provides an antibody having three different CDRs selectedfrom the group consisting of SEQ ID NO: 18, 19, and 20.

4. 2B5 Light Chain Sequences

Another embodiment provides an antibody or antigen binding fragmentthereof having a light chain encoded by a nucleic acid with at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to:

(SEQ ID NO: 21)

CACCTGCAAGGCCAGTCAGGATGTGGGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCTGAAGACTTGGCAGATTATTTCTG

Dashed underline represents the leader sequence. Single underlinerepresents the CDRs. Double underline represents the constant region.

The nucleic acid can be in a vector, for example an expression vector.The nucleic acid can be extrachromosal on inserted into a chromosome ofa host cell, for example a Chinese Hamster Ovary cell.

Another embodiment provides an antibody or antigen binding fragmentthereof having a light chain having an amino acid sequence with at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to:

(SEQ ID NO: 22)

Underline represents the leader sequence. Double underline representsCDRs. Dashed underline represents the constant region.

Another embodiment provides an antibody or antigen binding fragmentthereof having a light chain without the leader sequence having an aminoacid sequence with at least 50%, 60%, 70%. 80%. 85%. 90%. 95%. 99%, or100% sequence identity to:

(SEQ ID NO: 23)

Double underline represents CDRs. Dashed underline represents theconstant region.

The amino acid sequence for CDR1 of the 2B5 light chain is

(SEQ ID NO: 24) KASQDVSTAVA.

The amino acid sequence for CDR2 of the 2B5 light chain is

(SEQ ID NO: 13) WASTRHT.

The amino acid sequence for CDR3 of the 2B5 light chain is

(SEQ ID NO: 25) QQHYSTPWT.

One embodiment provides an antibody or antigen binding fragment thereofhaving a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%,99%, or 100% sequence identity to SEQ ID NO:22 or 23.

Another embodiment provides and antibody or antigen binding fragmentthereof having a light chain encoded by a nucleic acid with at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQID NO:21.

One embodiment provides an antibody having three different CDRs selectedfrom the group consisting of SEQ ID NO: 24, 13, and 25.

Another embodiment provides an antibody or antigen binding fragmentthereof having a heavy chain with an amino acid sequence having at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQID NO:16 or 17 and light chain with an amino acid sequence at least 50%,60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ IDNO:22 or 23, or light and heavy chain combinations thereof.

Another embodiment provides an antibody or antigen binding fragmentthereof having three different heavy chain CDRs with an amino acidselected from the group consisting of SEQ ID NOs:18, 19, and 20 andthree different light chain CDRs with amino acids selected from thegroup consisting of SEQ ID NOs: 24, 13, and 25.

6. 4G9 Heavy Chain Sequences

One embodiment provides a murine monoclonal antibody or antigen bindingfragment thereof isolated from hybridoma 4G9.

Another embodiment provides an antibody or antigen binding fragmentthereof having a heavy chain encoded by a nucleic acid with at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to:

(SEQ ID NO: 26)

TGGTACAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAACCTATGGAATGACCTGGGTGAAACAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACTCTGGAGTGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGAGGGGGACGGGGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCT

Underlined sequences correspond to complementarity determining regions(CDRs). Double underline sequence corresponds to the constant region.Dashed underlined sequences correspond to the leader sequence.

The nucleic acid can be in a vector, for example an expression vector.The nucleic acid can be extrachromosal on inserted into a chromosome ofa host cell, for example a Chinese Hamster Ovary cell.

One embodiment provides an antibody or antigen binding fragment thereofhaving heavy chain with an amino acid sequence having at least 50%, 60%,70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to:

(SEQ ID NO: 27)

Single underline corresponds to the leader sequence. Double under linecorresponds to CDRs, and dashed underline corresponds to the constantregion.

Another embodiment provides an antibody or antigen binding fragmentthereof having heavy chain without the leader sequence with an aminoacid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or100% sequence identity to:

(SEQ ID NO: 28)

Double under line corresponds to CDRs, and dashed underline correspondsto the constant region.

The amino acid sequence for CDR1 of the 4G9 heavy chain is

(SEQ ID NO: 29) TYGMT.

The amino acid sequence for CDR2 of the 4G9 heavy chain is

(SEQ ID NO: 30) WINTYSGVPTYADDFKG.

The amino acid sequence for CDR3 of the 4G9 heavy chain is

(SEQ ID NO: 31) GGRGFAY.

One embodiment provides a 4G9 antibody or antigen binding fragmentthereof having a heavy chain according to SEQ ID NO:27 or 28.

Another embodiment provides a 4G9 antibody or antigen binding fragmentthereof having a heavy chain encoded by a nucleic acid with at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQID NO:26.

One embodiment provides a 4G9 antibody having three different CDRsselected from the group consisting of SEQ ID NO: 29, 30, and 31.

7. 4G9 Light Chain Sequences

Another embodiment provides an antibody or antigen binding fragmentthereof having a light chain encoded by a nucleic acid with at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to:

(SEQ ID NO: 32)

TGCTGACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGCATTGGCACAAGCATACACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAGTAATAGCTGGCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG.

Dashed underline represents the leader sequence. Single underlinerepresents the CDRs. Double underline represents the constant region.

The nucleic acid can be in a vector, for example an expression vector.The nucleic acid can be extrachromosal on inserted into a chromosome ofa host cell, for example a Chinese Hamster Ovary cell.

Another embodiment provides an antibody or antigen binding fragmentthereof having a light chain having an amino acid sequence with at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to:

(SEQ ID NO: 33)

Underline represents the leader sequence. Double underline representsCDRs. Dashed underline represents the constant region.

Another embodiment provides an antibody or antigen binding fragmentthereof having a light chain without the leader sequence having an aminoacid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or100% sequence identity to:

(SEQ ID NO: 34)

Double underline represents CDRs. Dashed underline represents theconstant region.

The amino acid sequence for CDR1 of the 4G9 light chain is

(SEQ ID NO: 35) RASQSIGTSIH.

The amino acid sequence for CDR2 of the 4G9 light chain is

(SEQ ID NO: 36) YASESIS.

The amino acid sequence for CDR3 of the 4G9 light chain is

(SEQ ID NO: 37) QQSNSWPYT.

One embodiment provides a 4G9 antibody or antigen binding fragmentthereof having a light chain with at least 50%, 60%, 70%, 80%, 85%, 90%,95%, 99%, or 100% sequence identity to SEQ ID NO:33 or 34.

Another embodiment provides a 4G9 antibody or antigen binding fragmentthereof having a light chain encoded by a nucleic acid with at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQID NO:32.

One embodiment provides a 4G9 antibody having three different CDRsselected from the group consisting of SEQ ID NO:35, 36, and 37.

Another embodiment provides an antibody or antigen binding fragmentthereof having a heavy chain with an amino acid sequence having at least50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQID NO:27 or 28 and light chain with an amino acid sequence at least 50%,60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ IDNO:33 or 34 or light and heavy chain combinations thereof.

Another embodiment provides a 4G9 antibody or antigen binding fragmentthereof having three different heavy chain CDRs with an amino acidselected from the group consisting of SEQ ID NOs:29, 30, and 31 andthree different light chain CDRs with amino acids selected from thegroup consisting of SEQ ID NOs:35, 36, and 37.

D. PD-1 Activating Epitope Epitope-specific PD-1 binding moieties aredisclosed herein. In one embodiment, the disclosed binding moietiesimmunospecifically bind to PD-1, and activate PD-1 mediated signaltransduction.

The disclosed PD-1 epitope is formed by amino acids 96-110 of SEQ IDNO:1 and has an amino acid sequence as follows,

(SEQ ID NO: 38) TYLCGAISLAPKAQI.

1. Antibodies

One embodiment provides an antibody or epitope binding fragment thereofthat immunospecifically binds to SEQ ID NO:38 on the surface of animmune cell and activates the immune cell. In one embodiment, thepreferred immune cell is a T cell, more specifically a CD8⁺ T cell. Inanother embodiment, the antibody or epitope binding fragment thereofimmunospecifically binds SEQ ID NO:38 of PD-1 on the surface of animmune cell and promotes or induces an activating signal through PD-1 toactivate the immune cell.

The epitope specific antibody can be a monoclonal antibody, a humanizedantibody, a human antibody, a mouse antibody, a chimeric antibody, or afragment thereof. Exemplary antibodies and methods of their productionare discussed below.

2. Fusion Protein

In one embodiment, the epitope-specific PD-1 binding moiety is a fusionprotein. All of part of one or more of the disclosed PD-1 epitopeantibodies or epitope binding fragments above can be coupled to otherpolypeptides to form fusion proteins. Fusion polypeptides have a firstfusion partner including all or a part of one or more of the disclosedPD-1 epitope antibodies or epitope binding fragments fused to a secondpolypeptide directly or via a linker peptide sequence that is fused tothe second polypeptide. The fusion proteins optionally contain a domainthat functions to dimerize or multimerize two or more fusion proteins.The peptide/polypeptide linker domain can either be a separate domain,or alternatively can be contained within one of the other domains (firstpolypeptide or second polypeptide) of the fusion protein. Similarly, thedomain that functions to dimerize or multimerize the fusion proteins caneither be a separate domain, or alternatively can be contained withinone of the other domains (first polypeptide, second polypeptide orpeptide/polypeptide linker domain) of the fusion protein. In oneembodiment, the dimerization/multimerization domain and thepeptide/polypeptide linker domain are the same.

Fusion proteins disclosed herein are of formula I:

N—R₁—R₂—R₃—C

wherein “N” represents the N-terminus of the fusion protein, “C”represents the C-terminus of the fusion protein, “R₁” is all or part ofone of the disclosed PD-1 epitope antibodies or epitope bindingfragments, or functional variant or fragment thereof, “R₂” is anoptional peptide/polypeptide linker domain, and “R₃” is a secondpolypeptide. Alternatively, R₃ is all or part of one of the disclosedPD-1 epitope antibodies or epitope binding fragments, or functionalvariant or fragment thereof and R₁ is the second polypeptide.

Dimerization or multimerization can occur between or among two or morefusion proteins through dimerization or multimerization domains.Alternatively, dimerization or multimerization of fusion proteins canoccur by chemical crosslinking. The dimers or multimers that are formedcan be homodimeric/homomultimeric or heterodimeric/heteromultimeric.

3. Aptamers

In some embodiments, the epitope-specific binding moiety is an aptamer.Aptamers are molecules that interact with a target molecule, preferablyin a specific way. In one embodiment, the aptamer binds to SEQ ID NO:38on the surface of an immune cell and promotes or induces an activatingsignal through PD-1 to activate the immune cell. Typically aptamers aresmall nucleic acids ranging from 15-50 bases in length that fold intodefined secondary and tertiary structures, such as stem-loops orG-quartets. Aptamers can bind to protein, cells, small organicmolecules, or peptides. Aptamers can bind small molecules, such as ATPand theophiline, as well as large molecules, such as reversetranscriptase and thrombin. Aptamers can bind very tightly with Kd'sfrom the target molecule of less than 10-12 M. It is preferred that theaptamers bind the target molecule with a Kd less than 10-6, 10-8, 10-10,or 10-12. Aptamers can bind the target molecule with a very high degreeof specificity. For example, aptamers have been isolated that havegreater than a 10,000 fold difference in binding affinities between thetarget molecule and another molecule that differ at only a singleposition on the molecule. It is preferred that the aptamer have a Kdwith the target molecule at least 10, 100, 1000, 10,000, or 100,000 foldlower than the Kd with a background binding molecule. It is preferredwhen doing the comparison for a polypeptide for example, that thebackground molecule be a different polypeptide.

Representative examples of how to make and use aptamers to bind avariety of different target molecules are known in the art.

4. Small Molecules

In some embodiments, the epitope-specific binding moiety can be a smallmolecule. The term “small molecule” generally refers to small organiccompounds having a molecular weight of more than about 100 and less thanabout 2,500 Daltons, preferably between 100 and 2000, more preferablybetween about 100 and about 1250, more preferably between about 100 andabout 1000, more preferably between about 100 and about 750, morepreferably between about 200 and about 500 Daltons. Small moleculeagonists of the activating epitope of PD-1 can be identified usingroutine screening methods. In some embodiments, the screening assay caninclude random screening of large libraries of test compounds. Assayscan include determinations of PD-1 induced immune response or activationof T cells.

E. Pharmaceutical Compositions

Pharmaceutical compositions including the disclosed antibodies andantigen binding fragments thereof are provided. Pharmaceuticalcompositions containing the antibodies and antigen binding fragmentsthereof can be for administration by parenteral (intramuscular,intraperitoneal, intravenous (IV) or subcutaneous injection).

In some in vivo approaches, the compositions disclosed herein areadministered to a subject in a therapeutically effective amount. As usedherein the term “effective amount” or “therapeutically effective amount”means a dosage sufficient to treat, inhibit, or alleviate one or moresymptoms of the disorder being treated or to otherwise provide a desiredpharmacologic and/or physiologic effect. The precise dosage will varyaccording to a variety of factors such as subject-dependent variables(e.g., age, immune system health, etc.), the disease, and the treatmentbeing effected.

For the disclosed antibodies and antigen binding fragments thereof, asfurther studies are conducted, information will emerge regardingappropriate dosage levels for treatment of various conditions in variouspatients, and the ordinary skilled worker, considering the therapeuticcontext, age, and general health of the recipient, will be able toascertain proper dosing. The selected dosage depends upon the desiredtherapeutic effect, on the route of administration, and on the durationof the treatment desired. For the disclosed antibodies and antigenbinding fragments thereof, generally dosage levels of 0.001 to 20 mg/kgof body weight daily are administered to mammals. Generally, forintravenous injection or infusion, dosage may be lower.

In certain embodiments, the antibodies and antigen binding fragmentsthereof are administered locally, for example by injection directly intoa site to be treated. Typically, the injection causes an increasedlocalized concentration of the immunomodulatory agent composition whichis greater than that which can be achieved by systemic administration.The immunomodulatory agent compositions can be combined with a matrix asdescribed above to assist in creating an increased localizedconcentration of the polypeptide compositions by reducing the passivediffusion of the polypeptides out of the site to be treated.

1. Formulations for Parenteral Administration

In some embodiments, the compositions containing the disclosedantibodies and antigen binding fragments are administered in an aqueoussolution, by parenteral injection. The formulation may also be in theform of a suspension or emulsion. In general, pharmaceuticalcompositions are provided including effective amounts of an antibody orantigen binding fragment thereof, and optionally includepharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions optionallyinclude one or more for the following: diluents, sterile water, bufferedsaline of various buffer content (e.g., Tris-HCl, acetate, phosphate),pH and ionic strength; and additives such as detergents and solubilizingagents (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), andpreservatives (e.g., Thimersol, benzyl alcohol) and bulking substances(e.g., lactose, mannitol). Examples of non-aqueous solvents or vehiclesare propylene glycol, polyethylene glycol, vegetable oils, such as oliveoil and corn oil, gelatin, and injectable organic esters such as ethyloleate. The formulations may be lyophilized and redissolved/resuspendedimmediately before use. The formulation may be sterilized by, forexample, filtration through a bacteria retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions.

2. Formulations for Oral Administration

In some embodiments the antibody compositions are formulated for oraldelivery. The oral dosage forms of antibodies resist proteolysis and candeliver a greater fraction of immunoreactive antibody locally in thegastrointestinal tract for the treatment of infections or allow theabsorption of antibodies for the treatment or prevention of systemicconditions (Reilly, R M, et al. Clin Pharmacokinet., 32(4):313-23(1997); Victoria S Jasion and Bruce P Burnett, Nutr J.; 14: 22 (2015);and Philippart, M., et al., Drug Res (Stuttg) 66(03):113-120 (2016)).

Oral solid dosage forms are described generally in Remington'sPharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa.18042) at Chapter 89. Solid dosage forms include tablets, capsules,pills, troches or lozenges, cachets, pellets, powders, or granules orincorporation of the material into particulate preparations of polymericcompounds such as polylactic acid, polyglycolic acid, etc. or intoliposomes. Such compositions may influence the physical state,stability, rate of in vivo release, and rate of in vivo clearance of thedisclosed. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed.(1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which areherein incorporated by reference. The compositions may be prepared inliquid form, or may be in dried powder (e.g., lyophilized) form.Liposomal or proteinoid encapsulation may be used to formulate thecompositions. Liposomal encapsulation may be used and the liposomes maybe derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556).See also Marshall, K. In: Modern Pharmaceutics Edited by G. S. Bankerand C. T. Rhodes Chapter 10, 1979. In general, the formulation willinclude the peptide (or chemically modified forms thereof) and inertingredients which protect peptide in the stomach environment, andrelease of the biologically active material in the intestine.

The antibodies and antigen binding fragments thereof can be chemicallymodified so that oral delivery of the derivative is efficacious.Generally, the chemical modification contemplated is the attachment ofat least one moiety to the component molecule itself, where the moietypermits uptake into the blood stream from the stomach or intestine, oruptake directly into the intestinal mucosa. Also desired is the increasein overall stability of the component or components and increase incirculation time in the body. PEGylation is an exemplary chemicalmodification for pharmaceutical usage. Other moieties that may be usedinclude: propylene glycol, copolymers of ethylene glycol and propyleneglycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyproline, poly-1,3-dioxolane and poly-1,3,6-tioxocane[see, e.g., Abuchowski and Davis (1981) “Soluble Polymer-EnzymeAdducts,” in Enzymes as Drugs. Hocenberg and Roberts, eds.(Wiley-Interscience: New York, N.Y.) pp. 367-383; and Newmark, et al.(1982) J. Appl. Biochem. 4:185-189].

Another embodiment provides liquid dosage forms for oral administration,including pharmaceutically acceptable emulsions, solutions, suspensions,and syrups, which may contain other components including inert diluents;adjuvants such as wetting agents, emulsifying and suspending agents; andsweetening, flavoring, and perfuming agents.

For oral formulations, the location of release may be the stomach, thesmall intestine (the duodenum, the jejunem, or the ileum), or the largeintestine. In some embodiments, the release will avoid the deleteriouseffects of the stomach environment, either by protection of the agent(or derivative) or by release of the agent (or derivative) beyond thestomach environment, such as in the intestine. To ensure full gastricresistance a coating impermeable to at least pH 5.0 is essential.Examples of the more common inert ingredients that are used as entericcoatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D™, Aquateric™,cellulose acetate phthalate (CAP), Eudragit L™, Eudragit S™, andShellac™. These coatings may be used as mixed films.

3. Controlled Delivery Polymeric Matrices

The antibodies and antigen binding fragments thereof disclosed hereincan also be administered in controlled release formulations. Theantibodies and antigen binding fragments thereof can be incorporatedinto an inert matrix which permits release by either diffusion orleaching mechanisms, e.g., gums. Slowly degenerating matrices may alsobe incorporated into the formulation. Another form of a controlledrelease is based on the Oros therapeutic system (Alza Corp.), i.e., theantibody or antigen binding fragment thereof is enclosed in asemipermeable membrane which allows water to enter and push drug outthrough a single small opening due to osmotic effects.

Controlled release polymeric devices can be made for long term releasesystemically following implantation of a polymeric device (rod,cylinder, film, disk) or injection (microparticles). The matrix can bein the form of microparticles such as microspheres, where the agent isdispersed within a solid polymeric matrix or microcapsules, where thecore is of a different material than the polymeric shell, and thepeptide is dispersed or suspended in the core, which may be liquid orsolid in nature. Unless specifically defined herein, microparticles,microspheres, and microcapsules are used interchangeably. Alternatively,the polymer may be cast as a thin slab or film, ranging from nanometersto four centimeters, a powder produced by grinding or other standardtechniques, or even a gel such as a hydrogel.

Either non-biodegradable or biodegradable matrices can be used fordelivery of fusion polypeptides or nucleic acids encoding the fusionpolypeptides, although in some embodiments biodegradable matrices arepreferred. These may be natural or synthetic polymers, althoughsynthetic polymers are preferred in some embodiments due to the bettercharacterization of degradation and release profiles. The polymer isselected based on the period over which release is desired. In somecases linear release may be most useful, although in others a pulserelease or “bulk release” may provide more effective results. Thepolymer may be in the form of a hydrogel (typically in absorbing up toabout 90% by weight of water), and can optionally be cross-linked withmultivalent ions or polymers.

The matrices can be formed by solvent evaporation, spray drying, solventextraction and other methods known to those skilled in the art.Bioerodible microspheres can be prepared using any of the methodsdeveloped for making microspheres for drug delivery, for example, asdescribed by Mathiowitz and Langer, J. Controlled Release, 5:13-22(1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); andMathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988).

The devices can be formulated for local release to treat the area ofimplantation or injection—which will typically deliver a dosage that ismuch less than the dosage for treatment of an entire body—or systemicdelivery. These can be implanted or injected subcutaneously, into themuscle, fat, or swallowed.

III. Methods of Manufacture

A. Methods of Making Antibodies

The disclosed antibodies can be generated in cell culture, in phage, orin various animals, including but not limited to cows, rabbits, goats,mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys,chimpanzees, apes. In one embodiment, the various animals can betransgenic animals genetically engineered to produce human or humanizedantibodies. Therefore, in one embodiment, the antibody is a mammalianantibody. Phage techniques can be used to isolate an initial antibody orto generate variants with altered specificity or aviditycharacteristics. Such techniques are routine and well known in the art.In one embodiment, the antibody is produced by recombinant means knownin the art. For example, a recombinant antibody can be produced bytransfecting a host cell with a vector comprising a DNA sequenceencoding the antibody. One or more vectors can be used to transfect theDNA sequence expressing at least one VL and one VH region in the hostcell. Exemplary descriptions of recombinant means of antibody generationand production include Delves, Antibody Production: Essential Techniques(Wiley, 1997); Shephard, et al., Monoclonal Antibodies (OxfordUniversity Press, 2000); Goding, Monoclonal Antibodies: Principles AndPractice (Academic Press, 1993); Current Protocols In Immunology (JohnWiley & Sons, most recent edition).

The disclosed antibodies can be modified by recombinant means toincrease greater efficacy of the antibody in mediating the desiredfunction. Thus, it is within the scope of the invention that antibodiescan be modified by substitutions using recombinant means. Typically, thesubstitutions will be conservative substitutions. For example, at leastone amino acid in the constant region of the antibody can be replacedwith a different residue. See, e.g., U.S. Pat. Nos. 5,624,821,6,194,551, Application No. WO 9958572; and Angal, et al., Mol. Immunol.30:105-08 (1993). The modification in amino acids includes deletions,additions, and substitutions of amino acids. In some cases, such changesare made to reduce undesired activities, e.g., complement-dependentcytotoxicity. Frequently, the antibodies are labeled by joining, eithercovalently or non-covalently, a substance which provides for adetectable signal. A wide variety of labels and conjugation techniquesare known and are reported extensively in both the scientific and patentliterature. These antibodies can be screened for binding to proteins,polypeptides. See, e.g., Antibody Engineering: A Practical Approach(Oxford University Press, 1996).

For example, suitable antibodies with the desired biologic activitiescan be identified using in vitro assays including but not limited to:proliferation, migration, adhesion, soft agar growth, angiogenesis,cell-cell communication, apoptosis, transport, signal transduction, andin vivo assays such as the inhibition of tumor growth. The antibodiesprovided herein can also be useful in diagnostic applications. Ascapture or non-neutralizing antibodies, they can be screened for theability to bind to the specific antigen without inhibiting thereceptor-binding or biological activity of the antigen. As neutralizingantibodies, the antibodies can be useful in competitive binding assays.

Antibodies that can be used in the disclosed compositions and methodsinclude whole immunoglobulin (i.e., an intact antibody) of any class,fragments thereof, and synthetic proteins containing at least theantigen binding variable domain of an antibody. The variable domainsdiffer in sequence among antibodies and are used in the binding andspecificity of each particular antibody for its particular antigen.However, the variability is not usually evenly distributed through thevariable domains of antibodies. It is typically concentrated in threesegments called complementarity determining regions (CDRs) orhypervariable regions both in the light chain and the heavy chainvariable domains. The more highly conserved portions of the variabledomains are called the framework (FR). The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen binding site of antibodies. Also disclosed arefragments of antibodies which have bioactivity. The fragments, whetherattached to other sequences or not, include insertions, deletions,substitutions, or other selected modifications of particular regions orspecific amino acids residues, provided the activity of the fragment isnot significantly altered or impaired compared to the nonmodifiedantibody or antibody fragment.

Techniques can also be adapted for the production of single-chainantibodies based on the disclosed antibodies and antigen bindingfragments thereof. Methods for the production of single-chain antibodiesare well known to those of skill in the art. A single chain antibody canbe created by fusing together the variable domains of the heavy andlight chains using a short peptide linker, thereby reconstituting anantigen binding site on a single molecule. Single-chain antibodyvariable fragments (scFvs) in which the C-terminus of one variabledomain is tethered to the N-terminus of the other variable domain via a15 to 25 amino acid peptide or linker have been developed withoutsignificantly disrupting antigen binding or specificity of the binding.The linker is chosen to permit the heavy chain and light chain to bindtogether in their proper conformational orientation.

One embodiment provides divalent single-chain variable fragments(di-scFvs) that can be engineered by linking two scFvs. This can be doneby producing a single peptide chain with two VH and two VL regions,yielding tandem scFvs. ScFvs can also be designed with linker peptidesthat are too short for the two variable regions to fold together (aboutfive amino acids), forcing scFvs to dimerize. This type is known asdiabodies. Diabodies have been shown to have dissociation constants upto 40-fold lower than corresponding scFvs, meaning that they have a muchhigher affinity to their target. Still shorter linkers (one or two aminoacids) lead to the formation of trimers (triabodies or tribodies).Tetrabodies have also been produced. They exhibit an even higheraffinity to their targets than diabodies.

Another embodiment provides a monoclonal antibody obtained from asubstantially homogeneous population of antibodies, i.e., the individualantibodies within the population are identical except for possiblenaturally occurring mutations that may be present in a small subset ofthe antibody molecules. Monoclonal antibodies include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, as long as they exhibit thedesired antagonistic activity.

Monoclonal antibodies can be made using any procedure which producesmonoclonal antibodies. In a hybridoma method, a mouse or otherappropriate host animal is typically immunized with an immunizing agentto elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the immunizing agent.Alternatively, the lymphocytes may be immunized in vitro.

The disclosed antibodies may also be made by recombinant DNA methods.DNA encoding the disclosed antibodies can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). Libraries of antibodies oractive antibody fragments can also be generated and screened using phagedisplay techniques.

Methods of making antibodies using protein chemistry are also known inthe art. One method of producing proteins comprising the antibodies isto link two or more peptides or polypeptides together by proteinchemistry techniques. For example, peptides or polypeptides can bechemically synthesized using currently available laboratory equipmentusing either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc(tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., FosterCity, Calif.). One skilled in the art can readily appreciate that apeptide or polypeptide corresponding to the antibody, for example, canbe synthesized by standard chemical reactions. For example, a peptide orpolypeptide can be synthesized and not cleaved from its synthesis resinwhereas the other fragment of an antibody can be synthesized andsubsequently cleaved from the resin, thereby exposing a terminal groupwhich is functionally blocked on the other fragment. By peptidecondensation reactions, these two fragments can be covalently joined viaa peptide bond at their carboxyl and amino termini, respectively, toform an antibody, or fragment thereof. Alternatively, the peptide orpolypeptide is independently synthesized in vivo as described above.Once isolated, these independent peptides or polypeptides may be linkedto form an antibody or antigen binding fragment thereof via similarpeptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentsallow relatively short peptide fragments to be joined to produce largerpeptide fragments, polypeptides or whole protein domains. Alternatively,native chemical ligation of synthetic peptides can be utilized tosynthetically construct large peptides or polypeptides from shorterpeptide fragments. This method consists of a two-step chemical reaction.The first step is the chemoselective reaction of an unprotectedsynthetic peptide-alpha-thioester with another unprotected peptidesegment containing an amino-terminal Cys residue to give athioester-linked intermediate as the initial covalent product. Without achange in the reaction conditions, this intermediate undergoesspontaneous, rapid intramolecular reaction to form a native peptide bondat the ligation site.

B. Methods for Producing Isolated Nucleic Acid Molecules

One embodiment provides nucleic acids encoding the disclosed antibodiesor antigen binding fragments thereof. The nucleic acids can encode theentire antibody or antigen binding fragments thereof or a light chain,heavy chain, combinations thereof or CDRs thereof.

Isolated nucleic acid molecules can be produced by standard techniques,including, without limitation, common molecular cloning and chemicalnucleic acid synthesis techniques. For example, polymerase chainreaction (PCR) techniques can be used to obtain an isolated nucleic acidencoding a variant polypeptide. PCR is a technique in which targetnucleic acids are enzymatically amplified. Typically, sequenceinformation from the ends of the region of interest or beyond can beemployed to design oligonucleotide primers that are identical insequence to opposite strands of the template to be amplified. PCR can beused to amplify specific sequences from DNA as well as RNA, includingsequences from total genomic DNA or total cellular RNA. Primerstypically are 14 to 40 nucleotides in length, but can range from 10nucleotides to hundreds of nucleotides in length. General PCR techniquesare described, for example in PCR Primer: A Laboratory Manual, ed. byDieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995.When using RNA as a source of template, reverse transcriptase can beused to synthesize a complementary DNA (cDNA) strand. Ligase chainreaction, strand displacement amplification, self-sustained sequencereplication or nucleic acid sequence-based amplification also can beused to obtain isolated nucleic acids. See, for example, Lewis (1992)Genetic Engineering News 12:1; Guatelli et al. (1990) Proc. Natl. Acad.Sci. USA 87:1874-1878; and Weiss (1991) Science 254:1292-1293.

Isolated nucleic acids can be chemically synthesized, either as a singlenucleic acid molecule or as a series of oligonucleotides (e.g., usingphosphoramidite technology for automated DNA synthesis in the 3′ to 5′direction). For example, one or more pairs of long oligonucleotides(e.g., >100 nucleotides) can be synthesized that contain the desiredsequence, with each pair containing a short segment of complementarity(e.g., about 15 nucleotides) such that a duplex is formed when theoligonucleotide pair is annealed. DNA polymerase can be used to extendthe oligonucleotides, resulting in a single, double-stranded nucleicacid molecule per oligonucleotide pair, which then can be ligated into avector. Isolated nucleic acids can also obtained by mutagenesis.Protein-encoding nucleic acids can be mutated using standard techniques,including oligonucleotide-directed mutagenesis and/or site-directedmutagenesis through PCR. See, Short Protocols in Molecular Biology.Chapter 8, Green Publishing Associates and John Wiley & Sons, edited byAusubel et al, 1992.

IV. Methods of Use

The disclosed antibodies and antigen binding fragments thereof can beused to modulate an immune response in a subject in need thereof. Oneembodiment provides a method of activating immune cells expressing PD-1,for example T cells, to proliferate or enhance the biological activityof the immune cells expressing PD-1 by administering the disclosedantibodies and antigen fragments thereof, optionally including a secondtherapeutic agent.

A. Immune Response Stimulation

1. Therapeutic Strategies

Methods of inducing or enhancing an immune response in a subject areprovided.

Typically, the methods include administering a subject an effectiveamount of one or more of the disclosed antibodies and antigen bindingfragments thereof to immunospecifically bind to PD-1 and induce,promote, or enhance a stimulatory or activating signal through PD-1 toactivate the immune cell. The immune response can be, for exampleinducing, promoting or enhancing T cell activation, secretion ofcytokines by immune cells, T cell proliferation. The disclosedantibodies or antigen binding fragments thereof can be administered to asubject in need thereof in an effective amount to overcome T cellexhaustion and/or T cell anergy. Overcoming T cell exhaustion or T cellanergy can be determined by measuring T cell function using knowntechniques.

The methods can be used in vivo or ex vivo to induce, promote, orenhance a stimulating immune response.

In some embodiments, the antibody or antigen binding fragment thereof,or nucleic acid encoding the antibody or antigen binding fragmentthereof, is administered directly to the subject. In some embodiments,antibody or antigen binding fragment thereof is contacted with cells(e.g., immune cells) ex vivo, and the treat cells are administered tothe subject (e.g., adoptive transfer). The antibody or antigen bindingfragment thereof can enable a more robust immune response to bepossible. The disclosed compositions are useful to stimulate or enhanceimmune responses involving T cells causing an activating signal throughPD-1 on immune cells.

2. Subjects to be Treated

a. Treatment of Cancer

The disclosed antibodies and compositions thereof and methods can beused to treat cancer. Generally, the agents are used to stimulate orenhance an immune response to cancer in the subject by administering tothe subject an amount of a disclosed antibody or antigen bindingfragment thereof that induces, promotes, or enhances an activatingsignal through PD-1. The method can reduce one or more symptoms of thecancer.

The immune cells activated by the disclosed antibodies or fragmentsthereof can kill cancer cells and reduce tumor burden in a subject. Theterm “cancer cell” is meant to encompass both pre-malignant andmalignant cancer cells. In some embodiments, cancer refers to a benigntumor, which has remained localized. In other embodiments, cancer refersto a malignant tumor, which has invaded and destroyed neighboring bodystructures and spread to distant sites. In yet other embodiments, thecancer is associated with a specific cancer antigen (e.g., pan-carcinomaantigen (KS 1/4), ovarian carcinoma antigen (CA125), prostate specificantigen (PSA), carcinoembryonic antigen (CEA), CD19, CD20, HER2/neu,etc.).

The methods and antibody compositions disclosed herein are useful in thetreatment or prevention of a variety of cancers or other abnormalproliferative diseases, including (but not limited to) the following:carcinoma, including that of the bladder, breast, colon, kidney, liver,lung, ovary, pancreas, stomach, cervix, thyroid and skin; includingsquamous cell carcinoma; hematopoietic tumors of lymphoid lineage,including leukemia, acute lymphocytic leukemia, acute lymphoblasticleukemia, B-cell lymphoma, T-cell lymphoma, Berketts lymphoma;hematopoietic tumors of myeloid lineage, including acute and chronicmyelogenous leukemias and promyelocytic leukemia; tumors of mesenchymalorigin, including fibrosarcoma and rhabdomyoscarcoma; other tumors,including melanoma, seminoma, tetratocarcinoma, neuroblastoma andglioma; tumors of the central and peripheral nervous system, includingastrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin, including fibrosarcoma, rhabdomyoscarama, andosteosarcoma; and other tumors, including melanoma, xenodermapegmentosum, keratoactanthoma, seminoma, thyroid follicular cancer andteratocarcinoma.

Cancers caused by aberrations in apoptosis can also be treated by thedisclosed methods and compositions. Such cancers may include, but arenot be limited to, follicular lymphomas, carcinomas with p53 mutations,hormone dependent tumors of the breast, prostate and ovary, andprecancerous lesions such as familial adenomatous polyposis, andmyelodysplastic syndromes. In specific embodiments, malignancy ordysproliferative changes (such as metaplasias and dysplasias), orhyperproliferative disorders, are treated or prevented by the methodsand compositions in the ovary, bladder, breast, colon, lung, skin,pancreas, or uterus. In other specific embodiments, sarcoma, melanoma,or leukemia is treated or prevented by the methods and compositions.

Specific cancers and related disorders that can be treated or preventedby methods and compositions disclosed herein include, but are notlimited to, leukemias including, but not limited to, acute leukemia,acute lymphocytic leukemia, acute myelocytic leukemias such asmyeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemialeukemias and myelodysplastic syndrome, chronic leukemias such as butnot limited to, chronic myelocytic (granulocytic) leukemia, chroniclymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomassuch as, but not limited to, Hodgkin's disease or non-Hodgkin's diseaselymphomas (e.g., diffuse anaplastic lymphoma kinase (ALK) negative,large B-cell lymphoma (DLBCL); diffuse anaplastic lymphoma kinase (ALK)positive, large B-cell lymphoma (DLBCL); anaplastic lymphoma kinase(ALK) positive, ALK+ anaplastic large-cell lymphoma (ALCL), acutemyeloid lymphoma (AML)); multiple myelomas such as, but not limited to,smoldering multiple myeloma, nonsecretory myeloma, osteoscleroticmyeloma, plasma cell leukemia, solitary plasmacytoma and extramedullaryplasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy ofundetermined significance; benign monoclonal gammopathy; heavy chaindisease; bone and connective tissue sarcomas such as, but not limitedto, bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma,malignant giant cell tumor, fibrosarcoma of bone, chordoma, periostealsarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma;brain tumors including but not limited to, glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, nonglial tumor, acousticneurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including, but notlimited to, adenocarcinoma, lobular (small cell) carcinoma, intraductalcarcinoma, medullary breast cancer, mucinous breast cancer, tubularbreast cancer, papillary breast cancer, Paget's disease, andinflammatory breast cancer; adrenal cancer, including but not limitedto, pheochromocytom and adrenocortical carcinoma; thyroid cancer such asbut not limited to papillary or follicular thyroid cancer, medullarythyroid cancer and anaplastic thyroid cancer; pancreatic cancer,including but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers including but not limited to, Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers including, but not limited to, ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers, including, but not limited to, squamouscell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, includingbut not limited to, squamous cell carcinoma, melanoma, adenocarcinoma,basal cell carcinoma, sarcoma, and Paget's disease; cervical cancersincluding, but not limited to, squamous cell carcinoma, andadenocarcinoma; uterine cancers including, but not limited to,endometrial carcinoma and uterine sarcoma; ovarian cancers including,but not limited to, ovarian epithelial carcinoma, borderline tumor, germcell tumor, and stromal tumor; esophageal cancers including, but notlimited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma,mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma,plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma;stomach cancers including, but not limited to, adenocarcinoma, fungating(polypoid), ulcerating, superficial spreading, diffusely spreading,malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; coloncancers; rectal cancers; liver cancers including, but not limited to,hepatocellular carcinoma and hepatoblastoma, gallbladder cancersincluding, but not limited to, adenocarcinoma; cholangiocarcinomasincluding, but not limited to, papillary, nodular, and diffuse; lungcancers including but not limited to, non-small cell lung cancer,squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma,large-cell carcinoma and small-cell lung cancer; testicular cancersincluding, but not limited to, germinal tumor, seminoma, anaplastic,classic (typical), spermatocytic, nonseminoma, embryonal carcinoma,teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancersincluding, but not limited to, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers including, but not limitedto, squamous cell carcinoma; basal cancers; salivary gland cancersincluding, but not limited to, adenocarcinoma, mucoepidermoid carcinoma,and adenoidcystic carcinoma; pharynx cancers including, but not limitedto, squamous cell cancer, and verrucous; skin cancers including, but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers including, but notlimited to, renal cell cancer, adenocarcinoma, hypernephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor; bladder cancers including, but not limited to,transitional cell carcinoma, squamous cell cancer, adenocarcinoma,carcinosarcoma. In addition, cancers include myxosarcoma, osteogenicsarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma,synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma,bronchogenic carcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma and papillary adenocarcinomas (for areview of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997, InformedDecisions: The Complete Book of Cancer Diagnosis, Treatment, andRecovery, Viking Penguin, Penguin Books U.S.A., Inc., United States ofAmerica).

b. Treatment of Infections

The disclosed antibody compositions and methods can be used to treatinfections and infectious diseases. Generally, the agents are used tostimulate or enhance an immune response to an infection in the subjectby administering to the subject an amount of one or more of thedisclosed antibodies or antigen binding fragments thereof that sends aactivating or stimulating signal through PD-1. The method can reduce oneor more symptoms of the infection.

The infection or disease can be caused by a bacterium, virus, protozoan,helminth, or other microbial pathogen that enters intracellularly and isattacked, i.e., by cytotoxic T lymphocytes.

The infection or disease can be acute or chronic. An acute infection istypically an infection of short duration. During an acute microbialinfection, immune cells begin expressing immunomodulatory receptors.Accordingly, in some embodiments, the method includes increasing animmune stimulatory response against an acute infection.

The infection can be caused by, for example, but not limited to Candidaalbicans, Listeria monocytogenes, Streptococcus pyogenes, Streptococcuspneumoniae, Neisseria meningitidis, Staphylococcus aureus, Escherichiacoli, Acinetobacter baumannii, Pseudomonas aeruginosa or Mycobacterium.

In some embodiments, the disclosed antibody compositions are used totreat chronic infections, for example infections in which T cellexhaustion or T cell anergy has occurred causing the infection to remainwith the host over a prolonged period of time.

Exemplary infections to be treated are chronic infections cause by ahepatitis virus, a human immunodeficiency virus (HIV), a humanT-lymphotrophic virus (HTLV), a herpes virus, an Epstein-Barr virus, ora human papilloma virus.

Because viral infections are cleared primarily by T cells, an increasein T-cell activity would be therapeutically useful in situations wheremore rapid or thorough clearance of an infective viral agent would bebeneficial to an animal or human subject. Thus, the disclosedcompositions can be administered for the treatment of local or systemicviral infections, including, but not limited to, immunodeficiency (e.g.,HIV), papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza(e.g., human influenza virus A), and common cold (e.g., humanrhinovirus) and other viral infections, caused by, for example, HTLV,hepatitis virus, respiratory syncytial virus, vaccinia virus, and rabiesvirus. The molecules can be administered topically to treat viral skindiseases such as herpes lesions or shingles, or genital warts. Themolecules can also be administered systemically to treat systemic viraldiseases, including, but not limited to, AIDS, influenza, the commoncold, or encephalitis.

Representative infections that can be treated, include but are notlimited to infections cause by microorganisms including, but not limitedto, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio,Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium,Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus,Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus,Hemophilus influenza type B (HIB), Hyphomicrobium, Legionella,Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium,Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria,Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas,Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum,Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus,Thermoplasma, Thiobacillus, and Treponema, Vibrio, Yersinia,Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans,Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii,Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydialtrachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoebahistolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosomamansoni.

Other microorganisms that can be treated using the disclosedcompositions and methods include, bacteria, such as those of Klebsiella,Serratia, Pasteurella; pathogens associated with cholera, tetanus,botulism, anthrax, plague, and Lyme disease; or fungal or parasiticpathogens, such as Candida (albicans, krusei, glabrata, tropicalis,etc.), Cryptococcus, Aspergillus (fumigatus, niger, etc.), GenusMucorales (mucor, absidia, rhizophus), Sporothrix (schenkii),Blastomyces (dermatitidis), Paracoccidioides (brasiliensis),Coccidioides (immitis) and Histoplasma (capsulatuma), Entamoeba,histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp.,Giardia Zambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodiumvivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi,Toxoplasma gondi, etc.), Sporothrix, Blastomyces, Paracoccidioides,Coccidioides, Histoplasma, Entamoeba, Histolytica, Balantidium,Naegleria, Acanthamoeba, Giardia, Cryptosporidium, Pneumocystis,Plasmodium, Babesia, or Trypanosoma, etc.

V. Combination Therapies for Increasing Immune Responses

The disclosed antibodies and antigen binding fragments thereof andcompositions thereof can be administered to a subject in need thereofeither alone or in combination with one or more additional therapeuticagents. In some embodiments, the antibodies and antigen bindingfragments thereof and the additional therapeutic agent are administeredseparately, but simultaneously. The antibodies and antigen bindingfragments thereof and the additional therapeutic agent can also beadministered as part of the same composition. In other embodiments, theantibodies and antigen binding fragments thereof and the secondtherapeutic agent are administered separately and at different times,but as part of the same treatment regime. The additional therapeuticagents can be administered before, after, or in alternation with theadministration of the disclosed antibodies and antigen binding fragmentsthereof.

The subject can be administered a first therapeutic agent 1, 2, 3, 4, 5,6, or more hours, or 1, 2, 3, 4, 5, 6, 7, or more days beforeadministration of a second therapeutic agent. In some embodiments, thesubject can be administered one or more doses of the first agent every1, 2, 3, 4, 5, 6 7, 14, 21, 28, 35, or 48 days prior to a firstadministration of second agent. The antibodies and antigen bindingfragments thereof can be the first or the second therapeutic agent.

The antibodies and antigen binding fragments thereof and the additionaltherapeutic agent can be administered as part of a therapeutic regimen.For example, if a first therapeutic agent can be administered to asubject every fourth day, the second therapeutic agent can beadministered on the first, second, third, or fourth day, or combinationsthereof. The first therapeutic agent or second therapeutic agent may berepeatedly administered throughout the entire treatment regimen.

Exemplary additional therapeutic agents include, but are not limited to,cytokines, chemotherapeutic agents, radionuclides, otherimmunotherapeutics, enzymes, antibiotics, antivirals (especiallyprotease inhibitors alone or in combination with nucleosides fortreatment of HIV or Hepatitis B or C), anti-parasites (helminths,protozoans), growth factors, growth inhibitors, hormones, hormoneantagonists, antibodies and bioactive fragments thereof (includinghumanized, single chain, and chimeric antibodies), antigen and vaccineformulations (including adjuvants), peptide drugs, anti-inflammatories,ligands that bind to Toll-Like Receptors (including but not limited toCpG oligonucleotides) to activate the innate immune system, moleculesthat mobilize and optimize the adaptive immune system, other moleculesthat activate or up-regulate the action of cytotoxic T lymphocytes,natural killer cells and helper T-cells, and other molecules thatdeactivate or down-regulate suppressor or regulatory T-cells.

The additional therapeutic agents are selected based on the condition,disorder or disease to be treated. For example, the immunomodulatoryagent can be co-administered with one or more additional agents thatfunction to enhance or promote an immune response or reduce or inhibitan immune response.

A. Antimicrobials

In one embodiment, the antibodies and antigen binding fragments thereofcan be administered to the subject in combination with an antimicrobialsuch as an antibiotic, an antifungal, an antiviral, an anti-parasitics,or essential oil. In another embodiment, the disclosed antibodies andantigen binding fragments thereof can be used in a preventive orprophylactic role in the treatment and prevention of disease, and alsoin the context of severe trauma injuries like major burn, open bonefracture, accidental amputation or other wounds.

In some embodiments, the subject is administered the antibodies andantigen binding fragments thereof and/or the antimicrobial at time ofadmission to the hospital to prevent further bacterial, fungal or viralcomplications. The antibiotic can target pathogens and the antibodiesand antigen binding fragments thereof can stimulate the immune system toprovide an enhanced response to treat or prevent further infection ordisease.

1. Chemotherapeutic Agents

The antibodies and antigen binding fragments thereof can be combinedwith one or more chemotherapeutic agents and pro-apoptotic agents.Representative chemotherapeutic agents include, but are not limited toamsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin,docetaxel, doxorubicin, epirubicin, etoposide, fludarabine,fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide,irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin,lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin,mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin,procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil,temozolomide, teniposide, thiotepa, tioguanine, topotecan, treosulfan,vinblastine, vincristine, vindesine, vinorelbine, or a combinationthereof. Representative pro-apoptotic agents include, but are notlimited to fludarabinetaurosporine, cycloheximide, actinomycin D,lactosylceramide, 15d-PGJ(2) and combinations thereof.

2. Other Immunomodulators

a. PD-1 Antagonists

In some embodiments, the antibodies and antigen binding fragmentsthereof are co-administered with a PD-1 antagonist. Programmed Death-1(PD-1) is a member of the CD28 family of receptors that delivers anegative immune response when induced on T cells. Contact between PD-1and one of its ligands (B7-H1 or B7-DC) induces an inhibitory responsethat decreases T cell multiplication and/or the strength and/or durationof a T cell response. Suitable PD-1 antagonists are described in U.S.Pat. Nos. 8,114,845, 8,609,089, and 8,709,416, which are specificallyincorporated by reference herein in their entities, and includecompounds or agents that either bind to and block a ligand of PD-1 tointerfere with or inhibit the binding of the ligand to the PD-1receptor, or bind directly to and block the PD-1 receptor withoutinducing inhibitory signal transduction through the PD-1 receptor.

In some embodiments, the PD-1 receptor antagonist binds directly to thePD-1 receptor without triggering inhibitory signal transduction and alsobinds to a ligand of the PD-1 receptor to reduce or inhibit the ligandfrom triggering signal transduction through the PD-1 receptor. Byreducing the number and/or amount of ligands that bind to PD-1 receptorand trigger the transduction of an inhibitory signal, fewer cells areattenuated by the negative signal delivered by PD-1 signal transductionand a more robust immune response can be achieved.

It is believed that PD-1 signaling is driven by binding to a PD-1 ligand(such as B7-H1 or B7-DC) in close proximity to a peptide antigenpresented by major histocompatibility complex (MEW) (see, for example,Freeman, Proc. Natl. Acad. Sci. U. S. A, 105:10275-10276 (2008)).Therefore, proteins, antibodies or small molecules that preventco-ligation of PD-1 and TCR on the T cell membrane are also useful PD-1antagonists.

In some embodiments, the PD-1 receptor antagonists are small moleculeantagonists or antibodies that reduce or interfere with PD-1 receptorsignal transduction by binding to ligands of PD-1 or to PD-1 itself,especially where co-ligation of PD-1 with TCR does not follow suchbinding, thereby not triggering inhibitory signal transduction throughthe PD-1 receptor. Other PD-1 antagonists contemplated by the methods ofthis invention include antibodies that bind to PD-1 or ligands of PD-1,and other antibodies.

Suitable anti-PD-1 antibodies include, but are not limited to, thosedescribed in the following U.S. Pat. Nos. 7,332,582, 7,488,802,7,521,051, 7,524,498, 7,563,869, 7,981,416, 8,088,905, 8,287,856,8,580,247, 8,728,474, 8,779,105, 9,067,999, 9,073,994, 9,084,776,9,205,148, 9,358,289, 9,387,247, 9,492,539, 9,492,540, all of which areincorporated by reference in their entireties.

See also Berger et al., Clin. Cancer Res., 14:30443051 (2008).

Exemplary anti-PD-L1 antibodies include, but are not limited to, thosedescribed in the following U.S. Pat. Nos. 8,383,796, 9,102,725,9,273,135, 9,393,301, and 9,580,507 all of which are specificallyincorporated by reference herein in their entirety.

For anti-B7-DC (also referred to as anti-PD-L2) antibodies see U.S. Pat.Nos. 7,411,051, 7,052,694, 7,390,888, 8,188,238, and 9,255,147.

Other exemplary PD-1 receptor antagonists include, but are not limitedto PD-L2 polypeptides, including homologs and variants of these, as wellas active fragments of any of the foregoing, and fusion proteins thatincorporate any of these. In some embodiments, the fusion proteinincludes the soluble portion of B7-DC coupled to the Fc portion of anantibody, such as human IgG, and does not incorporate all or part of thetransmembrane portion of human B7-DC.

The PD-1 antagonist can also be a fragment of a mammalian PD-L1, forexample from mouse or primate, such as a human, wherein the fragmentbinds to and blocks PD-1 but does not result in inhibitory signaltransduction through PD-1. The fragments can also be part of a fusionprotein, for example an Ig fusion protein.

Other useful polypeptides PD-1 antagonists include those that bind tothe ligands of the PD-1 receptor. These include the PD-1 receptorprotein, or soluble fragments thereof, which can bind to the PD-1ligands, such as PD-L1 or B7-DC, and prevent binding to the endogenousPD-1 receptor, thereby preventing inhibitory signal transduction. PD-L1has also been shown to bind the protein B7.1 (Butte et al., Immunity,Vol. 27, pp. 111-122, (2007)). Such fragments also include the solubleECD portion of the PD-1 protein that includes mutations, such as theA99L mutation, that increases binding to the natural ligands (Molnar etal., PNAS, 105:10483-10488 (2008)). B7-1 or soluble fragments thereof,which can bind to the PD-L1 ligand and prevent binding to the endogenousPD-1 receptor, thereby preventing inhibitory signal transduction, arealso useful.

PD-1 and PD-L1 anti-sense nucleic acids, both DNA and RNA, as well assiRNA molecules can also be PD-1 antagonists. Such anti-sense moleculesprevent expression of PD-1 on T cells as well as production of T cellligands, such as PD-L1 and/or PD-L2. For example, siRNA (for example, ofabout 21 nucleotides in length, which is specific for the gene encodingPD-1, or encoding a PD-1 ligand, and which oligonucleotides can bereadily purchased commercially) complexed with carriers, such aspolyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest. 119(8):2231-2244 (2009), are readily taken up by cells that express PD-1 aswell as ligands of PD-1 and reduce expression of these receptors andligands to achieve a decrease in inhibitory signal transduction in Tcells, thereby activating T cells.

b. CTLA4 Antagonists

Other molecules useful in mediating the effects of T cells in an immuneresponse are also contemplated as additional therapeutic agents. In someembodiments, the molecule is an antagonist of CTLA4, for example anantagonistic anti-CTLA4 antibody. An example of an anti-CTLA4 antibodycontemplated for use in the methods of the invention includes anantibody as described in PCT/US2006/043690 (Fischkoff et al.,WO/2007/056539).

Dosages for anti-PD-1, anti-B7-H1, and anti-CTLA4 antibody, are known inthe art and can be in the range of, for example, 0.1 to 100 mg/kg, orwith shorter ranges of 1 to 50 mg/kg, or 10 to 20 mg/kg. An appropriatedose for a human subject can be between 5 and 15 mg/kg, with 10 mg/kg ofantibody (for example, human anti-PD-1 antibody) being a specificembodiment.

Specific examples of an anti-CTLA4 antibody useful in the methods of theinvention are Ipilimumab, a human anti-CTLA4 antibody, administered at adose of, for example, about 10 mg/kg, and Tremelimumab a humananti-CTLA4 antibody, administered at a dose of, for example, about 15mg/kg. See also Sammartino, et al., Clinical Kidney Journal,3(2):135-137 (2010), published online December 2009.

In other embodiments, the antagonist is a small molecule. A series ofsmall organic compounds have been shown to bind to the B7-1 ligand toprevent binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277:7363-7368(2002). Such small organics could be administered alone or together withan anti-CTLA4 antibody to reduce inhibitory signal transduction of Tcells.

3. Potentiating Agents

In some embodiments, additional therapeutic agents include apotentiating agent. The potentiating agent acts to increase efficacy theimmune response up-regulator, possibly by more than one mechanism,although the precise mechanism of action is not essential to the broadpractice of the present invention.

In some embodiments, the potentiating agent is cyclophosphamide.Cyclophosphamide (CTX, Cytoxan®, or Neosar®) is an oxazahosphorine drugand analogs include ifosfamide (IFO, Ifex), perfosfamide, trophosphamide(trofosfamide; Ixoten), and pharmaceutically acceptable salts, solvates,prodrugs and metabolites thereof (US patent application 20070202077which is incorporated in its entirety). Ifosfamide (MITOXANA®) is astructural analog of cyclophosphamide and its mechanism of action isconsidered to be identical or substantially similar to that ofcyclophosphamide. Perfosfamide (4-hydroperoxycyclophosphamide) andtrophosphamide are also alkylating agents, which are structurallyrelated to cyclophosphamide. For example, perfosfamide alkylates DNA,thereby inhibiting DNA replication and RNA and protein synthesis. Newoxazaphosphorines derivatives have been designed and evaluated with anattempt to improve the selectivity and response with reduced hosttoxicity (Liang J, Huang M, Duan W, Yu X Q, Zhou S. Design of newoxazaphosphorine anticancer drugs. Curr Pharm Des. 2007; 13(9):963-78.Review). These include mafosfamide (NSC 345842), glufosfamide (D19575,beta-D-glucosylisophosphoramide mustard), S-(−)-bromofosfamide (CBM-11),NSC 612567 (aldophosphamide perhydrothiazine) and NSC 613060(aldophosphamide thiazolidine). Mafosfamide is an oxazaphosphorineanalog that is a chemically stable 4-thioethane sulfonic acid salt of4-hydroxy-CPA. Glufosfamide is IFO derivative in which theisophosphoramide mustard, the alkylating metabolite of IFO, isglycosidically linked to a beta-D-glucose molecule. Additionalcyclophosphamide analogs are described in U.S. Pat. No. 5,190,929entitled “Cyclophosphamide analogs useful as anti-tumor agents” which isincorporated herein by reference in its entirety.

While CTX itself is nontoxic, some of its metabolites are cytotoxicalkylating agents that induce DNA crosslinking and, at higher doses,strand breaks. Many cells are resistant to CTX because they express highlevels of the detoxifying enzyme aldehyde dehydrogenase (ALDH). CTXtargets proliferating lymphocytes, as lymphocytes (but not hematopoieticstem cells) express only low levels of ALDH, and cycling cells are mostsensitive to DNA alkylation agents.

In one embodiment low doses of CTX are used in combination with thedisclosed antibodies and antigen binding fragments thereof. Low doses ofCTX (<200 mg/kg) can have immune stimulatory effects, includingstimulation of anti-tumor immune responses in humans and mouse models ofcancer (Brode & Cooke Crit Rev. Immunol. 28:109-126 (2008)). These lowdoses are sub-therapeutic and do not have a direct anti-tumor activity.In contrast, high doses of CTX inhibit the anti-tumor response. Severalmechanisms may explain the role of CTX in potentiation of anti-tumorimmune response: (a) depletion of CD4+CD25+FoxP3+Treg (and specificallyproliferating Treg, which may be especially suppressive), (b) depletionof B lymphocytes; (c) induction of nitric oxide (NO), resulting insuppression of tumor cell growth; (d) mobilization and expansion ofCD11b+Gr-1+MDSC. These primary effects have numerous secondary effects;for example following Treg depletion macrophages produce more IFN-γ andless IL-10. CTX has also been shown to induce type I IFN expression andpromote homeostatic proliferation of lymphocytes.

Treg depletion is most often cited as the mechanism by which CTXpotentiates the anti-tumor immune response. This conclusion is based inpart by the results of adoptive transfer experiments. In the AB1-HAtumor model, CTX treatment at Day 9 gives a 75% cure rate. Transfer ofpurified Treg at Day 12 almost completely inhibited the CTX response(van der Most et al. Cancer Immunol. Immunother. 58:1219-1228 (2009). Asimilar result was observed in the HHD2 tumor model: adoptive transferof CD4+CD25+Treg after CTX pretreatment eliminated therapeutic responseto vaccine (Taieb, J. J. Immunol. 176:2722-2729 (2006)).

Numerous human clinical trials have demonstrated that low dose CTX is asafe, well-tolerated, and effective agent for promoting anti-tumorimmune responses (Bas, & Mastrangelo Cancer Immunol. Immunother. 47:1-12(1998)).

In one embodiment, the optimal dose for CTX to potentiate an anti-tumorimmune response, is one that lowers overall T cell counts by loweringTreg levels below the normal range but is subtherapeutic (see Machielset al. Cancer Res. 61:3689-3697 (2001)).

In some embodiments, CTX is used as an immunopotentiating agent at adose of 300 mg/m². In another embodiment, for an average male (6 ft, 170pound (78 kg) with a body surface area of 1.98 m²), 300 mg/m², the doseof CTX is 8 mg/kg, or 624 mg of total protein. In mouse models ofcancer, efficacy has been seen at doses ranging from 15-150 mg/kg, whichrelates to 0.45-4.5 mg of total protein in a 30 g mouse (Machiels et al.Cancer Res. 61:3689-3697 (2001), Hengst et al Cancer Res. 41:2163-2167(1981), Hengst Cancer Res. 40:2135-2141 (1980)).

For larger mammals, such as a primate, such as a human, patient, suchmg/m² doses may be used but unit doses administered over a finite timeinterval may also be used. Such unit doses may be administered on adaily basis for a finite time period, such as up to 3 days, or up to 5days, or up to 7 days, or up to 10 days, or up to 15 days or up to 20days or up to 25 days, are all specifically contemplated by theinvention. The same regimen may be applied for the other potentiatingagents recited herein.

In other embodiments, the potentiating agent is an agent that reducesactivity and/or number of regulatory T lymphocytes (T-regs), such asSunitinib (SUTEN®), anti-TGFβ or Imatinib (GLEEVAC®). The recitedtreatment regimen may also include administering an adjuvant.

Useful potentiating agents also include mitosis inhibitors, such aspaclitaxol, aromatase inhibitors (e.g. Letrozole) and angiogenesisinhibitors (VEGF inhibitors e.g. Avastin, VEGF-Trap) (see, for example,Li et al., Vascular endothelial growth factor blockade reducesintratumoral regulatory T cells and enhances the efficacy of aGM-CSF-secreting cancer immunotherapy. Clin Cancer Res. 2006 Nov. 15;12(22):6808-16), anthracyclines, oxaliplatin, doxorubicin, TLR4antagonists, and IL-18 antagonists.

VI. Transgenic Animals

One embodiment provides a transgenic animal that produces antibodies orantigen binding fragments thereof having heavy chain CDRs with an aminoacid sequences according to SEQ ID NOs:6, 7, and 8 and light chain CDRswith amino acids according to SEQ ID NOs: 12, 13, and 14. In oneembodiment, the transgenic animal is a rodent, for example a mouse.Another embodiment provides a transgenic animal that produces antibodiesor antigen binding fragments thereof having heavy chain CDRs with anamino acid sequences according to SEQ ID NO:18, 19, and 20 and threedifferent light chain CDRs with amino acids selected from the groupconsisting of SEQ ID NOs: 24, 13, and 25. In one embodiment, thetransgenic animal is a rodent, for example a mouse.

Another embodiment provides a transgenic animal that produces antibodiesor antigen binding fragments thereof having heavy chain CDRs with anamino acid sequences according to SEQ ID NOs: 29, 30, and 31 and lightchain CDRs with amino acids according to SEQ ID NOs: 35, 36, and 37. Inone embodiment, the transgenic animal is a rodent, for example a mouse.

Methods of making transgenic animals that produce antibodies are knownin the art. See for example A. Jakobovits, Curr Opin Biotechnol.,6(5):561-6 (1995) and Bruggemann, M., et al., Arch Immunol Ther Exp(Warsz)., 63(2):101-108 (2015); Jakobovits, A., et al., “From XenoMousetechnology to panitumumab, the first fully human antibody product fromtransgenic mice.” Nat Biotechnol. 25(10):1134-43 (2007); Lonberg N.(2005) “Human antibodies from transgenic animals.” Nat Biotechnol.23(9):1117-25 and U.S. Pat. Nos. 9,708,635; 9,686,970; 9,499,838;9,445,581; 9,388,446; 8,835,712; 8,703,485; 8,232,449; 7,795,494; and5,939,598.

EXAMPLES Example 1: Anti-PD-1 Antibody Production

Results

Production of anti-PD-1 antibodies produced clones 4G9, 4C12, and 5C2which were selected for characterization.

Example 2: Interaction Kinetics Between Anti-PD-1 Antibodies and PD-1

Materials and Methods

Antibodies from clones 4G9, 4C12, and 5C2 where characterized using aBiocore™ system available from GE. The analyte was mouse or human PD-1and the ligand was the anti-PD-1 antibody. Analyte concentrations were0, 62.5, 125, 250, 500, and 1000 nM where indicated.

Results

FIG. 1 and Table 1 show interaction analysis of 4G9 with human PD-1. Theequilibrium association constant (K_(A)) was 9.52×10⁵ (1/M). Theequilibrium dissociation constant (K_(D)) was 1.05×10⁻⁶ (M).

TABLE 1 Interaction analysis of 4G9 with human PD-1. Ka Kd KA KD ConcLigand Analyte (1/Ms) (1/s) Rmax (1/M) (M) (Nm) Chi² 4G9 Human NA NA15.1 9.52 × 10⁵ 1.05 × 10⁻⁶ 0 0.464 580 RU PD-1 125 250 500 500 1000

FIG. 2 and Table 2 show interaction analysis of 4G9 with mouse PD-1. Theequilibrium association constant (K_(A)) was 1.94×10⁵ (1/M). Theequilibrium dissociation constant (K_(D)) was 5.15×10⁻⁶ (M).

TABLE 2 Interaction analysis of 4G9 with mouse PD-1. Ka Kd KA KD ConcLigand Analyte (1/Ms) (1/s) Rmax (1/M) (M) (Nm) Chi² 4G9 Human 2.00 ×10³ 0.0103 38.8 1.94 × 10⁵ 5.15 × 10⁻⁶ 0 0.112 580 RU PD-1 125 250 500500 1000

FIG. 3 and Table 3 show interaction analysis of 4C12 with human PD-1.The equilibrium association constant (K_(A)) was 3.14×10⁶ (1/M). Theequilibrium dissociation constant (K_(D)) was 3.19×10⁻⁷ (M).

TABLE 3 Interaction analysis of 4C12 with human PD-1. Ka Kd KA KD ConcLigand Analyte (1/Ms) (1/s) Rmax (1/M) (M) (Nm) Chi² 4C12 Human NA NA33.9 3.14 × 10⁶ 3.19 × 10⁻⁷ 0 2.33 425 RU PD-1 125 250 500 500 1000

FIG. 4 and Table 4 show interaction analysis of 5C2 with human PD-1. Theequilibrium association constant (K_(A)) was 2.02×10⁵ (1/M). Theequilibrium dissociation constant (K_(D)) was 4.95×10⁻⁶ (M).

TABLE 4 Interaction analysis of 5C2 with human PD-1. Ka Kd KA KD ConcLigand Analyte (1/Ms) (1/s) Rmax (1/M) (M) (Nm) Chi² 5C2 Human NA NA28.4 2.02 × 10⁵ 4.95 × 10⁻⁶ 0 0.0263 320 RU PD-1 125 250 500 500 1000

FIG. 5 and Table 5 show interaction analysis of 5C2 with mouse PD-1. Theequilibrium association constant (K_(A)) was 1.18×10⁶ (1/M). Theequilibrium dissociation constant (K_(D)) was 8.50×10⁻⁷ (M).

TABLE 5 Interaction analysis of 5C2 with mouse PD-1. Ka Kd KA KD ConcLigand Analyte (1/Ms) (1/s) Rmax (1/M) (M) (Nm) Chi² 5C2 Human NA NA 111.18 × 10⁶ 8.50 × 10⁻⁷ 0 0.107 320 RU PD-1 125 250 500 500 1000

Example 3: Binding of Anti-PD-1 Antibodies to EL4 Cells

Materials and Methods

Murine EL4 cells that constitutively express PD-1 were used influorescence activated cell sorter to evaluate binding of 4G9, 5C2, and4C12 to the cells.

Results

FIG. 6A is a flow cytometry histogram that shows commercial anti-PD-1binds to EL4 cells and the control isotype antibody does not bind to EL4cells. FIG. 6B is a flow cytometry histogram that shows that 4G9, 5C2,and 4C12 bind to EL4 cells and secondary antibody alone does not. FIG.6C is a flow cytometry histogram that shows that 4G9 anti-PD-1 antibodybinds to EL4 cells and secondary antibody alone does not. FIG. 6D is aflow cytometry histogram that shows that 5C2 anti-PD-1 antibody binds toEL4 cells and secondary antibody alone does not. FIG. 6E is a flowcytometry histogram that shows that 4C12 anti-PD-1 antibody binds to EL4cells and secondary antibody alone does not.

Example 4: Agonistic Activity of Anti-PD-1 Antibodies

Materials and Methods

Mouse CD4 T Cells

Purified mouse CD4 T cells were stimulated with anti-CD3/anti-CD28 Abfor 48 hrs, then anti-PD-1 Abs (10 ug/ml) were added to culture alongwith Protein A beads for another 48 hrs. In some samples anti-PD-L1 Abwas added to block PD-1/PD-L1 interaction to dissect the agonist effectof test Abs. CBA assay was used to detect IFNγ and IL-2 concentrationsin supernatants.

Human CD4 T Cells

Purified human CD4 T cells were stimulated with anti-CD3/anti-CD28 Abfor 48 hrs, then anti-PD-1 Abs (1 or 10 ug/ml) were added to culturealong with Protein A beads for another 48 hrs. CBA assay was used todetect IFNγ concentrations in supernatants.

Results

FIGS. 7A and 7B show that stimulated CD4 T cells treated with anti-PD-1antibodies 5C2, 4C12, and 4G9, had higher concentrations of IFNγ andIL-2 in supernatants compared to untreated cells or cells treated withanti-PD-L1 antibodies. FIG. 7C shows that stimulated human CD4 T cellstreated with anti-PD-1 antibodies 4G9 and 5C2 had a higher concentrationof IFNγ supernatant compared to untreated cells or cells treated withisotype control antibody.

Example 5: Hybridomas Enhance Akt Phosphorylation

Materials and Methods

Intracellular staining of pAKT (S473) was used as a marker for T cellactivation.

Results

Hybridomas from mice immunized with peptide E enhanced thephosphorylation of Akt (S473) in mouse CD4 T cells (FIG. 8).

Example 6: Characterization of Three Anti-PD-1 Antibodies

Materials and Methods

Purified antibodies from hybridomas 5C2, 4C12, and 4G9 werecharacterized.

Results

The isotypes for each of the three anti-PD-1 antibodies were determined.5C2 and 4C12 were both IgG1 isotype and 4G9 was found to be IgG2bisotype (FIG. 9). Hybridoma sequencing showed 100% sequence identity for5C2 and 4C12 antibodies.

Example 7: 4G9 and 5C2 Specifically Bind to Human PD-1

Materials and Methods:

An ELISA assay was used to determine binding of 4G9 and 5C2 to humanPD-1-Fc.

Results:

FIG. 10 shows the results of an ELISA assay to evaluate binding of 4G9and 5C2 antibodies to human PD-1-Fc. 4G9 and 5C2 both specifically bindto human PD-1. FIG. 11 A is a flow cytometry histogram showing that 4G9and 5C2 do not bind PD-1 KO CD4 T cells, while they do bind CD4 T cellsfrom wild-type mice (FIG. 11B).

Example 8: Signaling

Materials and Methods:

Mouse CD4 T cells were pre-stimulated for 48 hours to ensure PD-1expression then treated with purified 4G9 and 5C2 antibodies. Theconcentration of pS6 was determined using an ELISA kit (Cell SignalingTech).

Results:

In contrast to blocking antibodies (RMP1-14 and J43), 4G9 and 5C2activate T cells through S6 pathway. Treatment of 48 h pre-stimulated(to ensure PD-1 expression) mouse CD4 T cells with purified 4G9 and 5C2antibodies led to significant increase of pS6 within the linear range(ELISA kit Cell Signaling Tech, *P<0.05, **P<0.01, ***P<0.001 comparedto Untreated). This phenomenon is due to a direct activation rather thanPD-1/PD-L1 blockade since treatment with Commercial Ab-1 (RMP1-14) andAb-2 (J43) that blocks PD-1/PD-L1 interaction does not result in pS6increase.

Example 8: In Vivo Efficacy Evaluation

Materials and Methods:

FIG. 13A is a schematic illustration of the TC-1 tumor model used inthis experiment. Briefly, mice were subcutaneously injected with TC-1tumor cells at day 0. At day 10 (D10), day 17 (D17), and day 24 (D24)after tumor injection, mice were treated with vaccine (E7+PADRE+Quil A).Mice were treated with anti-PD-1 antibodies at day 10, day 14, day 17,day 21, day 24, and day 28.

Results:

Anti-PD-1 antibodies 4G9, 4C12, and 5C2 reduced tumor volume andincreased survival when combined with E7 vaccine (FIG. 13B-13D).

Example 9: In Vivo Efficacy Evaluation-Anti-EpE Antibodies

Materials and Methods:

Antibodies against epitope E were generated and tested in the TC-1 tumormodel as described in Example 8 above and FIG. 14A.

Results:

Mice treated with 4G9, an antibody generated against epitope E,exhibited significantly lower tumor volume and higher survival rate whencompared to traditional checkpoint inhibitors, anti-PD1 blockingantibodies RMP1-14 and J43 (FIG. 14B-14C).

1.-44. (canceled)
 45. A method of inducing cellular activity in animmune cell that expresses programmed cell death receptor protein(PD-1), comprising contacting the immune cell with an antibody orantigen-binding fragment thereof that binds to an epitope of PD-1,wherein the epitope comprises SEQ ID NO:
 38. 46. The method of claim 45,wherein the antibody or antigen-binding fragment thereof comprises: a.the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within anyone of the heavy chain variable region (HCVR) sequences selected fromthe group consisting of SEQ ID NOs: 27 or 28; and the three light chainCDRs (LCDR1, LCDR2 and LCDR3) contained within any one of the lightchain variable region (LCVR) sequences selected from the groupconsisting of SEQ ID NOs: 33 or 34; b. the HCDR1, HCDR2 and HCDR3contained within any one of the HCVR sequences selected from the groupconsisting of SEQ ID NOs: 4 or 5; and the LCDR1, LCDR2 and LCDR3contained within any one of the LCVR sequences selected from the groupconsisting of SEQ ID NOs: 10 or 11; or c. the HCDR1, HCDR2 and HCDR3contained within any one of the HCVR sequences selected from the groupconsisting of SEQ ID NOs: 16 or 17; and the LCDR1, LCDR2 and LCDR3contained within any one of the LCVR sequences selected from the groupconsisting of SEQ ID NOs: 22 or
 23. 47. The method of claim 45, whereinthe cellular activity comprises cell proliferation.
 48. The method ofclaim 45, wherein the cellular activity comprises cytokine activation.49. The method of claim 45, wherein the immune cell is a T cell.
 50. Themethod of claim 49, wherein the T cell is a CD8 T cell.
 51. The methodof claim 46, wherein the antibody or antigen-binding fragment thereofcomprises: (a) an HCDR1 domain having an amino acid sequence of SEQ IDNO: 29; (b) an HCDR2 domain having an amino acid sequence of SEQ ID NO:30; (c) an HCDR3 domain having an amino acid sequence of SEQ ID NO: 31;(d) an LCDR1 domain having an amino acid sequence of SEQ ID NO: 35; (e)an LCDR2 domain having an amino acid sequence of SEQ ID NO: 36; and (0an LCDR3 domain having an amino acid sequence of SEQ ID NO:
 37. 52. Themethod of claim 46, wherein the antibody or antigen-binding fragmentthereof comprises: (a) an HCDR1 domain having an amino acid sequence ofSEQ ID NO: 6; (b) an HCDR2 domain having an amino acid sequence of SEQID NO: 7; (c) an HCDR3 domain having an amino acid sequence of SEQ IDNO: 8; (d) an LCDR1 domain having an amino acid sequence of SEQ ID NO:12; (e) an LCDR2 domain having an amino acid sequence of SEQ ID NO: 13;and (0 an LCDR3 domain having an amino acid sequence of SEQ ID NO: 14.53. The method of claim 46, wherein the antibody or antigen-bindingfragment thereof comprises: (a) an HCDR1 domain having an amino acidsequence of SEQ ID NO: 18; (b) an HCDR2 domain having an amino acidsequence of SEQ ID NO: 19; (c) an HCDR3 domain having an amino acidsequence of SEQ ID NO: 20; (d) an LCDR1 domain having an amino acidsequence of SEQ ID NO: 24; (e) an LCDR2 domain having an amino acidsequence of SEQ ID NO: 13; and (f) an LCDR3 domain having an amino acidsequence of SEQ ID NO:
 25. 54. An isolated antibody or antigen-bindingfragment thereof that binds specifically to PD-1, wherein the antibodycomprises the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) containedwithin any one of the heavy chain variable region (HCVR) sequencesselected from the group consisting of SEQ ID NOs:16 or 17; and the threelight chain CDRs (LCDR1, LCDR2 and LCDR3) contained within any one ofthe light chain variable region (LCVR) sequences selected from the groupconsisting of SEQ ID NOs: 22 or
 23. 55. The antibody or antigen-bindingfragment thereof of claim 54, comprising: (a) an HCDR1 domain having anamino acid sequence of SEQ ID NO: 18; (b) an HCDR2 domain having anamino acid sequence of SEQ ID NO: 19; (c) an HCDR3 domain having anamino acid sequence of SEQ ID NO: 20; (d) an LCDR1 domain having anamino acid sequence of SEQ ID NO: 24; (e) an LCDR2 domain having anamino acid sequence of SEQ ID NO: 13; and (0 an LCDR3 domain having anamino acid sequence of SEQ ID NO:
 25. 56. A pharmaceutical compositioncomprising the antibody or antibody binding fragment thereof accordingto claim
 54. 57. The pharmaceutical composition of claim 56, furthercomprising a second therapeutic agent.
 58. The pharmaceuticalcomposition of claim 57, further comprising a pharmaceuticallyacceptable excipient.
 59. The pharmaceutical composition of claim 58,wherein the second therapeutic agent is a chemotherapeutic agent.
 60. Amethod of inducing, promoting, or enhancing an immune response in asubject in need thereof, comprising, administering to the subject aneffective amount of the isolated antibody or antigen binding fragment ofclaim 54, or the pharmaceutical composition of claim 56, to induce,promote, or enhance an immune response in the subject.
 61. A method forreducing tumor burden in a subject in need thereof, comprisingadministering to the subject an effective amount of the isolatedantibody or antigen binding fragment of claim 54, or the pharmaceuticalcomposition of claim 56 to reduce tumor burden in the subject.
 62. Themethod of claim 61, wherein the tumor burden is from a bladder tumor,breast tumor, colorectal tumor, kidney tumor, liver tumor, lung tumor,ovary tumor, pancreas tumor, stomach tumor, cervical tumor, thyroidtumor, hematopoietic tumor of lymphoid lineage, hematopoietic tumor ofmyeloid lineage, tumor of the central and peripheral nervous system, ortumor of mesenchymal origin.
 63. A method for treating an infection in asubject in need thereof, comprising, administering to the subject aneffective amount of the antibody or antigen binding fragment of claim54, or the pharmaceutical composition of claim 56, to treat theinfection in the subject.
 64. An isolated antibody or antigen-bindingfragment thereof that binds specifically to PD-1, wherein the antibodycomprises: (a) an HCDR1 domain having an amino acid sequence of SEQ IDNO: 6; (b) an HCDR2 domain having an amino acid sequence of SEQ ID NO:7; (c) an HCDR3 domain having an amino acid sequence of SEQ ID NO: 8;(d) an LCDR1 domain having an amino acid sequence of SEQ ID NO: 12; (e)an LCDR2 domain having an amino acid sequence of SEQ ID NO: 13; and (f)an LCDR3 domain having an amino acid sequence of SEQ ID NO:
 14. 65. Apharmaceutical composition comprising the antibody or antibody bindingfragment thereof according to claim
 64. 66. The pharmaceuticalcomposition of claim 65, further comprising a second therapeutic agent.67. The pharmaceutical composition of claim 66, further comprising apharmaceutically acceptable excipient.
 68. The pharmaceuticalcomposition of claim 67, wherein the second therapeutic agent is achemotherapeutic agent.
 69. A method of inducing, promoting, orenhancing an immune response in a subject in need thereof, comprising,administering to the subject an effective amount of the isolatedantibody or antigen binding fragment of claim 64, or the pharmaceuticalcomposition of claim 65, to induce, promote, or enhance an immuneresponse in the subject.
 70. A method for reducing tumor burden in asubject in need thereof, comprising administering to the subject aneffective amount of the isolated antibody or antigen binding fragment ofclaim 64, or the pharmaceutical composition of claim 65 to reduce tumorburden in the subject.
 71. The method of claim 70, wherein the tumorburden is from a bladder tumor, breast tumor, colorectal tumor, kidneytumor, liver tumor, lung tumor, ovary tumor, pancreas tumor, stomachtumor, cervical tumor, thyroid tumor, hematopoietic tumor of lymphoidlineage, hematopoietic tumor of myeloid lineage, tumor of the centraland peripheral nervous system, or tumor of mesenchymal origin.
 72. Amethod for treating an infection in a subject in need thereof,comprising, administering to the subject an effective amount of theantibody or antigen binding fragment of claim 64, or the pharmaceuticalcomposition of claim 65, to treat the infection in the subject.